- By Jingwei Sun A THESIS Submitted to Michigan State University i n partial fulfillment of the requirements f or the degree of Food Science Master of Science 2015 ABSTRACT - By Jingwei Sun Plant phenolic compounds have seen increased interest within the field of cereal science in the recent decade, indicated by the ongoing research in their functional and health properties. The objectives of this thesis were (1) to quantify the total phenoli c acid and ferulic acid contents of selected Michigan grown wheat varieties harvested in 2013 and 2014, and (2) to compare differences in post - processing antioxidant retention and baking properties between cookies fortified with flour using four different antioxidant formulations (20% bran, 4.4% insoluble arabinoxylan powder, ferulic acid powder, or 20% digested bran with ferulic acid powder). The total phenolic acid contents ranged from 3.6 to 6.8 mg GAE/g for the 2013 crop year, and 4.0 to 6.0 mg GAE/g fo r the 2014 crop year. The total ferulic acid contents ranged from 272 ± 12 g/g to 412 ± 1 g/g for the 2013 crop year , and from 316 ± 36 g/g to 467 ± 34 g/g for the 2014 crop year. The ratios of the averaged total phenolic acid contents in the milled fractions of bran, shorts, and flour for all varieties were 5:4:1. For the baking analysis, the 20% bran blend showed the least amount of ferulic acid degradation after baking among the flour blends studied, with a 90% retention rate, and is the recommended form for ferulic acid fortification. Copyright by JINGWEI S UN 2015 ACKNOWLE DGEMENTS I would like to thank my advisor Dr. Ng for his vast knowledge in the cereal scien ce field and his willingness to help me with many questions and difficulties with my experiments, and for his patience and understanding, and I would like to thank Dr. Strasburg and Dr. Olson for assisting with my project as Guidance C ommittee M embers. TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ............................... vi i LIST OF FIGURES ................................ ................................ ................................ .............................. ix KEY TO ABBREVIATIONS ................................ ................................ ................................ .................. x i C HAPTER 1 INTRODUCTION ................................ ................................ ................................ ............................... 1 CHAPTER 2 LITERATURE REVIEW ................................ ................................ ................................ ....................... 4 2.1 . O verview of F erulic A cid ................................ ................................ ................................ ....... 4 2.2 . Health Benefits of Ferulic Acid ................................ ................................ ............................ 7 2.3 . Factors Affecting Antioxidant Contents in Different Wheat Varieties ................................ . 9 2.4 . Bioavailability of Phenolic Acids ................................ ................................ ......................... 10 2.5 . Extraction of Phenolic Acids ................................ ................................ ............................... 11 2.6 . Effects of Storage and Processing on Phenolic Acid Contents ................................ ........... 14 2.7 . Commercial Feasibility ................................ ................................ ................................ ....... 15 Ch HAPTER 3 MATERIALS AND METHODS ................................ ................................ ................................ .......... 17 3.1. Material s ................................ ................................ ................................ ............................. 17 3 .1 .1. Wheat kernel S amples ................................ ................................ ................................ . 17 3.1.2. Chemicals ................................ ................................ ................................ ..................... 20 3 . 2. Methods ................................ ................................ ................................ ............................. 20 3 . 2 .1 . Milling of Wheat Fractions and Ba s ic Wheat Characterization ................................ .. 20 3.2.2.Experimental Outline of Antioxidant Quantification Analysis ................................ .... 22 3 . 2 .3 . Extraction of Phenolic Acids by Alkaline Digestion ................................ ..................... 24 3.2 .4 . Extraction of Ferulic Acid by Enzymatic Digestion ................................ ...................... 25 3 . 2 .5 . Quantification of Wheat Flour Total Phenolic Contents by Colorimetric Analysis ..... 28 3.2 .6 . Quantification of Wheat Flour Ferulic Acid Content by Reverse - Phase High - Performance Liquid Chromatography ................................ ................................ ................... 28 3.2.7 . Ferulic Acid Stability during Cookie Baking ................................ ................................ . 30 3.3. Data analysis: ANOVA ................................ ................................ ................................ ......... 33 CHAPTER 4 RESULTS AND DISCUSSION ................................ ................................ ................................ ........... 34 4 .1 . Moisture, Ash, and Protein Contents and Soundness of Wheat Samples ......................... 34 4 .2 . Total Phenolic Content (TPC) of Wheat Lines ................................ ................................ ... 39 4.2.1 . Total Phenolic Content (TPC) of Whole Ground Flour ................................ ................ 39 4.2.2.Total Phenolic Content of Milling Fractions of Wheat Varieties ................................ . 48 4.3 . Ferulic Acid Content of Whole Ground Flour ................................ ................................ ..... 52 4.4 . Pearson Correlations of the Phenolic and Ferulic Acid Contents of Studied Varieties ...... 58 4.5 . Effect of Crop Year on Ferulic Acid Content of Whole Ground Flour Samples our ........... 60 4.6 . Baking Quality of Cookies after Fortification with Free and Bound Forms of Ferulic Aci d 64 4.6.1 . Visual Quality of Cookies Baked from Flour Fortified with Free and Bound Forms of Ferulic Acid ................................ ................................ ................................ ............................ 64 4.6.2 . Effect of Ferulic Acid Source on Cookie Moisture Contents ................................ ....... 66 4.6.3 . Effect of Ferulic Acid Source on Cookie Color ................................ ............................. 68 4.6.4 . Cookie Diameter and H eight ................................ ................................ ....................... 71 4.6.5 . Cookie H ardness ................................ ................................ ................................ .......... 74 4.6.6 . Changes in T otal Ferulic Acid C ontents after Baking ................................ ................... 7 4 CHAPTER 5 ................................ ................................ ..... APPENDICES ................................ ................................ ................................ ................................ .. 82 A PPENDIX A . Supplementary Data for Wheat Phenolic Quantification and Cookie Baking .... 83 APPENDIX B . Viability of Enzymatic Digestion of Wheat Flour with Feruloyl Esterase ............ 87 APPENDIX C . Statistical Analysis Raw Data . ................................ ................................ ............. 92 APPENDIX D . Colorimetric Analysis Raw Data ................................ ................................ ........ 129 ................................ ................................ ................................ ....................... LI ST OF TABLES T able 1. Name Listing of MSU Soft Wheat Varieties from 2013 and 2014 Crop Years Selected for Testing ................................ ................................ ................................ ................................ .......... 1 8 ............................... ................................ ................................ ......................... Table 3 . b. Moisture, Ash, and Protein Contents of Whole Ground Flour from Michigan Wheat Varieties from the 2014 Harvest (n=3) ................................ ................................ ........................ 37 Ta ble 4 . Ferulic Acid Contents of Select 2013 MSU Wheat Breeding Varieties .......................... 5 3 Table 5 . Ferulic Acid Contents of Select 2014 MSU Wheat Breeding Varieties .......................... 5 5 Table 6 . Select Environmenta l C onditions of Growing Locations in East - Central Michigan from the harvesting and planting seasons of 2013 to 2014 (MAWN, 2012,2013,2014) ...................... 63 Table 7 . Moisture Contents of Cookies Baked From Different Ambassador Wheat Flour Blends ................................ ................................ ................................ ................................ ....................... 6 7 Table 8 . Color Analysis of Ferulic Acid - Fortified Cookies made from Amba ssador flour ............ 69 Table 9 . Physical Qualities of Cookies Baked from Different Blends of Ambassador Wheat Flour Fortified with Identical Amounts of Ferulic Acid (n=3) ................................ .............................. 73 Table 1 0 . Preparation of ferulic acid standard solution for RP - HPLC analysis ............................ 83 Table 11 . Pear s on Product - Moment Correlation Coefficient between Different P arameters from C ookie B aking ................................ ................................ ................................ ............................... 8 4 Table 12 . Signific ant D ifferences of Total P opulations measured us ing O ne way ANOVA with Fis her C omparison. Populations with p<0.05 were deemed significantly different ................... 8 5 Table 13.1 . Raw Data for 2013 Total Ferulic Acid Content ................................ .......................... 93 Table 13 .2. Raw Data for 2013 Bound Ferulic Acid Content ................................ ........................ 96 Table 13 .3. Raw Data for 2013 Free Ferulic Acid Content ................................ ............................ 99 Table 13 .4. Raw Data for 2014 Total Ferulic Acid Content ................................ ......................... 102 Table 13 .5. Raw Data for 2014 Bound Ferulic Acid Content ................................ ..................... 105 Table 13 .6. Raw Data for 2014 Free Ferulic Acid Content ................................ .......................... 108 Table 13 .7. Raw Data for 2013 Total Phenolic Acid Content ................................ ..................... 111 Table 13 .8. Raw Data for 2013 Bound Phenolic Content ................................ .......................... 114 Table 13 .9. Raw Data for 2013 Free Phenolic Content ................................ ............................... 117 Table 13 .10. Raw Data for 2014 Total Phenolic Content ................................ ........................... 120 Table 13 .11. Raw Data for 2014 Bound Phenolic Content ................................ ........................ 123 Table 13 .12. Raw Data for 2014 Free Phenolic Content ................................ ............................ 126 Table 14 .1. Phenolic Acid Contents of Selected 2013 MSU Wheat Breeding Varieties ............. 1 29 Table 14 .2. Phenolic Acid Contents of Selected 2014 MSU Wheat Breeding Varieties ............ 131 Table 14 .3. Total Phenolic Acid Con tents of 2013 Wheat Fractions ................................ .......... 132 Table 14 .4. Total Phenolic Acid Contents of 2014 Wheat Fractions ................................ .......... 134 LI ST OF FIGURES Figure 1. Structure of Ferulic Acid (Srinivasan et al., 2001) ................................ ............................ 6 ................................ ..... ................................ ................................ ................................ ................................ ........ Figure 4 .a. Total Phenolic Contents of Wheat Varieties from the 2013 MSU Wheat Breeding Program. Variety numbers and corresponding names are listed in Table 1. Error bars represent one standard deviation of measurements in triplicate. Significant groupings were analyzed with nd fully presented in Appendix C, Table 13 . ................................ ................................ ................................ ................................ ....................... 42 Figure 4 .b. Bou nd Phenolic Contents of Varieties from the 2013 MSU Wheat Breeding Program. Variety numbers and corresponding names are listed in Table 1. Error bars represent one standard deviation of measurements in triplicate. Significant groupings were analyzed with and fully presented in Appendix C , Table 13 . Bound phenolic content is obtained by subtracting the free from the total phenolic contents. 4 3 Figure 4 .c. Free Phenolic Contents of Lines from the 2013 MSU Wheat Breeding Program. Variety numbers and corresponding names are listed in Table 1. Error bars represent one standard deviation of measurements in triplicate. Significant groupings were analyzed with and fully presented in Appendix C, Table 13 . ................................ ................................ ................................ ................................ ....................... 44 Figure 5 .a. Total Phenolic Contents of 2014 MSU Wheat Breeding Varieties. Variety numbers and corresponding names are listed in Table 1. Error bars represent one standard deviation of Significant Difference test (p<0.05) and fully presented in Appendix C, Table 13 . . ................................ ...... 45 Figure 5 .b. Bound Phenolic Contents of 2014 MSU Wheat Breeding varieties. Variety numbers and corresponding names are listed in Table 1. Error bars represent one stand ard deviation of Difference test (p<0.05) a nd fully presented in Appendix C , Table 13 . Bound phenolic content is obtained by subtracting the free from the total phenolic contents. ................................ .......... 4 6 Figure 5 .c. Free Phenolic Contents of 2014 MSU Wheat Breeding varieties. Error bars represent one standard deviation of measurements in triplicate. Significant groupings were analyzed with (p<0.05) a nd fully presented in Appendix C , Table 13 . 47 Figure 6 . Total Phenolic Contents of Milled Wheat Fractions from S elected Michigan wheat varieties harvested in 2013. Samples were analyzed in triplicate and results are reported as mean values with one standard deviation. ................................ ................................ .................. 5 0 Figure 7 . Total Phenolic Contents of Milled Wheat Fractions from S elected Michigan Wheat Varieties harvested in 2014. Samples were analyzed in triplicate and reported as mean values with one standard deviation. ................................ ................................ ................................ ........ 5 1 Fig ure 8 . Representative Visual Appearance of Cookies baked using Different Flour Blends Consisting of white flour and: (a) No additions, (b) 20% bran, (c) 4.4% arabinoxylan, (d) Free FA powder, and (e) Digest ed 20% bran with free FA powder ................................ .......................... 6 5 Figure 9 . Total Ferul ic Acid Content of Baked Cookie S amples made from Various White Flour Blends. Bars not sharing the same letter are significantly different from each other (p<0.05). The blue line is an indication of the initially fortified level mea sured in the cookie doug h of the treatments before baking, excluding the original control flour . The ferulic acid content of the control cookie without fortification wa s included for reference. ................................ ................................ ................................ ................................ ...................... 7 8 Figure 10 . Percent of Ferulic Acid Retained in Cookies after Baking Cookie Doughs made from Four Different White Flour Blends of Wheat Variety Ambassador. ................................ ............ 7 9 Figure 1 1. UV - Visible S pectrum of Ferulic Acid Extracted from Michigan S oft Wheat Whole Ground Flour (Ambassador) . ................................ ................................ ................................ ........ 86 Figure 1 2. Ferulic Content of Wheat Sa mples Treated with Constant Xylanase at 20 U and increasing amounts of Feruloyl Esterase (see Table 2, without pre - heating treatment). ........... 88 Figure 1 3. Ferulic Acid Content of Wheat S amples Treated with Constant FE at 20 U and Increasing Concentrations of Xyl (Table 2, with hea ting). ................................ ........................... 9 0 Figure 1 4. Ferulic Acid Content of Wheat S amples Treated With Constant Levels of Xyl at 20 U, and Increasing Additions of FE, With Heat Pre - T reatment . ................................ ......................... 9 1 KEY TO ABBREVIATIONS ABTS: 2,2' - azino - b is(3 - ethylbenzothiazoline - 6 - sulphonic acid) DPPH: 2,2 - diphenyl - 1 - picrylhydrazyl FA: Ferulic acid FE: Feruloyl esterase GA: Gallic acid GAE: Gallic Acid Equivalent HPLC: High Performance Liquid Chromatography MAWN: Michigan Automated Weather Network ORAC S: Oxygen radical absorbance capacity PPO: Polyphenol o xidase TPC: Total Phenolic Contents TFA: Trifluoroacetic acid PC: Phenolic Contents Xyl: Xylanase CHAPTER 1 INTRODUCTION Cereal grains contain a wide variety of antioxidants, including phenolic acids, flavonoids, and condensed tannins (Dykes and Rooney, 2007). Phenolic compounds are not as widely studied as other antioxidants such as vitamins A, C, and E, but they are highly abundant in nature, and are essential components in many plant structures. In the past decade there has been increasing interest in phenolic compounds due to their potential benefits to human health. Studies on these compounds have indicate d protective effects when ingested in sufficient quantities (Dillard and German, 2000). Bound phenolic acids, such as ferulic acid, have been found to have greater bioavailability than free ferulic acids (Ro n dini et al., 2004). The majority of ferulic ac id in wheat flour is in its bound form, but many of the existing antioxidant fortification studies using ferulic acid used free soluble ferulic acid powder, and did not mention potential differences in processing properties between bound vs free ferulic ac ids (Ou et al., 2005; Koh and Ng 2008). During food processing such as baking, bound ferulic acid in wheat flour may be more stable and experience less loss than its free form, being less available for reaction with the other food ingredients. This means t hat it may not be applicable to use the results of experiments utilizing free ferulic acid powder to interpret effects on naturally - occurring bound ferulic acid existing in plant products. Free ferulic acid is also more accessible to yeast and other microo rganisms, which may metabolize it and break it down to inactive components, decreasing the overall antioxidant capability of a flour (Huang et al., 1993). It is therefore important to conduct experiments that isolate bound and free ferulic acids and observ e their individual effects on human nutrition and food processing. There are new food product development opportunities in promoting higher levels of such intrinsic antioxidants for nutritional value and health benefits, although current U.S. regulations d o not permit labeling claims for any antioxidants nor any phenolic acids. To be eligible to claim the health benefits reported by nutritional and medical studies (Sultana et al., 2005 ; Kroon et al., 1997), the bioactive antioxidants require standardization of effective dosages. Many by - products of food processing are rich in phenolic compounds. Examples (DDGS) from biofuel production (Ezeji and Blaschek, 2008). The se products may serve as sources of abundant raw material for commercial phenolic acid production and fortification of foods . Quantifying the antioxidant capacity and ferulic acid content of current wheat varieties and future lines can lead to understanding of the diversity of wheat antioxidants. Michigan primarily produces soft red winter (SRW) and soft white winter (SWW) wheats, and there are research programs focusing on the development of new soft wheat lines. Soft white winter wheat is the main focus of research in this thesis. The antioxidant and ferulic acid contents of Michigan - grown soft wheat have not been determined, and survey eff orts of antioxidant capacity and ferulic acid content among SRW and SWW wheat samples from the MSU breeding program is warranted. Screening for wheat varieties with the genetic potential for high antioxidant content yields would provide the Michigan wheat farming and milling sectors the ability to produce new food ingredients and products containing higher antioxidant contents as adaptation for shifting consumer preferences and market demands. The minimum physiologically effective dose is yet to be agreed upon. In cooperation with ongoing current research on the health benefits of phenolic acids, the phenolic acid contents of Michigan wheat varieties should be surveyed to assist in determining the amounts of phenolic acids naturally present in wheat for fut ure reference. Quantification of Michigan wheat antioxidant content will assist wheat breeding programs in selecting for varieties with different antioxidant yields. The objectives of this study were to: 1) Determine the contents of bound and free phenolic and ferulic acid s of selected Michigan - grown soft wheat varieties. 2) Compare the differences in post - processing antioxidant retention of bound ferulic acid and free ferulic acid, and the baking qualities of cookies made with different flour formulations fortified with the same level of antioxidants (bound ferulic acid or free ferulic acid). - - - - - Figure 1. Structure of Ferulic Acid (Srinivasan et al., 2001). Figure 2 . Resonance Stabilization of Ferulic Acid (Srinivasan et al., 2001). - - - - - - - - - - - - - - - - 2.5 . Extraction of Phenolic Acids - - - 2.6 . Effects of Storage and Processing on Phenolic Acids Content - - - - - - - - - - - - - - - - - - - - - - - Wheat kernels Whole ground flour Milling fractions Bran Shorts Flour Buhler mill - - - - - - - - - - - - - - - - - - - 3.3. Da ta A nalysis: ANOVA - - CHAPTER 4 RESULTS AND DISCUSSION 4.1. Moisture, Ash, and Protein Contents and Soundness of Wheat Samples The whole ground flour of the wheat varieties harvested in 2013 and 2014 were tested for their moisture, as h, protein contents, and falling number. The whole ground flour was prepared as mentioned in 3.1.1. All wheat varieties displayed normal ranges of moisture, ash and protein contents that are typical of the values found in commercially used soft wheat flour. (Table 3 a, b ) . Most of the wheat varieties were sound, having Falling Number values above the 25 0 second threshold, indicating little to no pre - sprouting. (Table 3 a, b ). There were slight increases for the 2014 year in protein content in many of the flour samples, but the overall differences between the two years was not significantly different when measured using paired t - test , with a p - value of 0.065. Table 3 . a. Moisture, Ash, and Protein Contents of Whole Ground Flour from Michigan Wheat Varieties from the 2013 Harvest (n=3) Table 3 . b. Moisture, Ash, and Protein Contents of Whole Ground Flour from Michigan Wheat Varieties from the 2014 Harvest (n=3) 4.2 . Total Phenolic Content (TPC) of Wheat Lines A preliminary comparison between enzymatic and alkaline digestion s was performed (Appendix B ) , and alkaline digestion was found to be more efficient and convenient, and was used for all phenolic extractions for all types of wheat samples for the rest of the current study. The total, bound, and free phenolic contents of the 2013 and 2014 wheat varieties harvested for this study were measured by the Folin - Ciocalteu assay us ing a gallic acid standard for comparison. T he colorimetric analysis was used to take into account the cumulative effects of the total combined reducing compounds found in the whole ground flour and the milled fractions, and the specificity of ferulic acid as the main phenolic acid constituent in the wheat samples was measured by RP - HPLC analysis discussed in later sections. 4.2.1 . Total Phenolic Content (TPC) of Whole Ground Wheat Flour The TPC, bound PC and free PC levels of whole ground flour of the 2013 and 2014 varieties from the MSU Wheat Breeding Program selected for this thesis study were examined and expressed as milligrams of gallic acid equiva lent (GAE) per gram of whole ground flour (Figures 4 and 5 ). There were significantly different groups of varieties with noticeable low and high group ranges. The number of significant ly different groups decreased from the 2013 to the 2014 harvest for both the bound and free phenolic measurements , alongside an overall increase in TPC in the 2014 harvest , as could be seen in their higher population F value and lower population p value (Appendix A, Table 11 ) . G allic acid is a popular reference compound used as the standard for TPC colorimetric assays due to its solubility in water, non - volatility, and stability when exposed to pH and temperature changes. From the 2013 harvested wheat, varieties with the highest total PC were varieties #34 (F2003 ) and # 52 (Jupiter) at 6.0 and 6.9 mg GAE/g , respectively , and those with the lowest were varieties #17 (F2037) and #6 ( F1047 ) (Figure 4 .a) at 3.6 and 3.7 mg GAE/g , respectively . The varieties with the highest bound PCs were #1 (F1014) and #52 (Jupiter) at 4.6 an d 5.3 mg GAE/g respectively , while those with the lowest were #9 (F1049) at 1.5 mg GAE/g and # 17 (F20 37 ) at 1.4 mg GAE/g (Figure 4 .b ). For the free PC (i.e., water soluble), the highest values were found for #9 (F1049) and #32 (F2011 ), while the lowest wer e found for #18 (F2035), #45 (F2021), and #52 (Jupiter) (Figure 4 .c ). In general, the samples with relatively higher bound PCs had relatively lower free PCs and vice versa, but such a trend was not observed for all studied varieties. It is also noticeable that the free phenolic range levels were on a much smaller scale th an those of the bound fraction. Therefore, the level of bound PC or TPC may be more important for breeding, since the amounts of free phenolic acids in wheat were much smaller . Among the 2014 varieties, there were 34 lines repeated from the 2013 crops, w hile 5 lines were new entries. The lowest TPC value was found for variety F2037 (#17) at 4.0 mg GAE/g and the highest was for Unnamed 1 (#59) at 6.0 mg GAE/g (Figure 5 .a). The measured bound and free PC levels of the 39 wheat varieties from 2014 also displ ayed noticeable differences. For bound PC, the greatest measured value was for F2022 (#43) at 4.9 mg GAE/g and the lowest was for Ambassador (#51) at 2.8 mg GAE/g ( Figure 5 .b). The highest free PC value was from Aubrey (#47) at 1.7mg GAE/g , and the lowest was from F2022 at 0.8 mg GAE/g (#43). There was no clear correlation (r = - 0.1275) between the measured free and bound PC values of the combined years . Similar to 2013 varieties, some 2014 varieties with relatively high bound PC values had relatively low f ree PC values (varieties #38 and #43), and some of the varieties with relatively low bound PC values had relatively high free PC values (varieties #51 and #58) ( Figure 5 .c). Although the TPC values were similar between the 2013 and 2014 harvests, 2014 vari eties generally had higher bound PC values and lower free PC values than those of the 2013 varieties . The TPC values obtained in this current study were higher than results from other published studies in which the sample preparation for total ferulic aci d content analysis was used for TPC assay (Adom et al., 2003). Adom and colleagues reported bound PC values between 0.8 to 1.5 mg GAE/g, which was significantly lower than those in the current study. Besides the very different wheat genetic traits and grow ing locations, sample preparation procedure was also not identical. In the current study, samples were extracted with water or 2N NaOH followed by neutralization, and the supernatant was used for TPC assay directly, while in the Adom et al. study, the sup ernatant of NaOH - digested wheat extracted for HPLC analysis was used for TPC analysis for speed and convenience. Their supernatant s contained less total reducing materials available for colorimetric reactions (Adom et al, 2003), presumably a result of some loss of phenolic compounds due to the extraction process. Differences in extraction time may also have led to different colorimetric readings. Adom and colleagues (Adom et al., 2003) utilized a digestion time of 1 hour, but the current experiment could no t attain complete digestion of the whole ground flour until 4 hours. Figure 4 .a. Total P henolic C ontents of W heat V arieties from the 2013 MSU Wheat Breeding Program. Variety numbers and corresponding names are listed in Table 1 . Error bars represent one standard deviation of measurements in triplicate. Significant groupings were analyzed with cant Difference test (p<0.05) a nd fully presented in Appendix C , Table 13 . 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 17 6 9 38 10 5 16 41 53 4 54 57 40 56 15 1 23 20 37 42 31 19 8 11 14 30 28 50 36 Total Phenolic Content (mg GAE/g) 2013 Variety Number 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 39 48 18 13 3 35 12 49 22 29 26 32 25 7 27 45 55 51 43 33 21 24 47 2 44 46 34 52 Total Phenolic Content (mg GAE/g) 2013 Variety Number Figure 4 .b. Bound P henolic C ontents of Varieties from the 2013 MSU Wheat Breeding Program. Variety numbers and corresponding names are listed in Table 1. Error bars represent one standard deviation of measurements in triplicate. Significant groupings were analyzed with nce test (p<0.05) and fully presented in Appendix C , Table 13 . Bound phenolic content is obtained by subtracting the free from the total phenolic contents. 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 17 9 38 10 41 57 16 6 30 8 40 4 56 20 36 54 23 27 42 1 19 15 37 39 53 29 51 2 55 Bound Phenolic Content (mg GAE/g) 2013 Variety Number 0.00 1.00 2.00 3.00 4.00 5.00 6.00 28 12 3 35 49 11 13 31 7 32 14 33 26 50 43 22 24 25 48 21 47 18 5 45 44 34 46 52 Bound Phenolic Content (mg GAE/g) 2013 Variety Number Figure 4 .c. Free P henolic C ontents of L ines from the 2013 MSU Wheat Breeding Program. Variety nu mbers and corresponding names are listed in Table 1. Error bars represent one standard deviation of measurements in triplicate. Significant groupings were analyzed with nd fully presented in Appendix C, Table 13 . 0.0 0.5 1.0 1.5 2.0 2.5 3.0 45 18 52 14 21 11 10 23 22 28 53 48 37 35 15 54 31 2 25 6 33 34 7 39 16 4 19 27 Free Phenolic Content (mg GAE/g) 2013 Variety Number 0.0 0.5 1.0 1.5 2.0 2.5 3.0 43 30 12 5 29 1 13 51 50 20 42 3 38 17 47 8 26 24 57 49 41 44 55 40 56 46 36 32 9 Free Phenolic Content (mg GAE/g) 2013 Variety Number Figure 5 .a. Total P henolic C ontents of 2014 MSU Wheat Breeding V arieties. Variety numbers and corresponding names are listed in Table 1. Error bars represent one standard deviation of measurements in triplicate. Significant groupings were Difference test (p<0.05) a nd fully presented in Appendix C , Table 13 . 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 17 51 58 30 31 62 47 40 27 12 61 9 37 45 24 46 34 21 5 33 Total Phenolic Content (mg GAE/g) 2014 Variety Number 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 41 13 8 6 14 52 60 42 38 39 29 23 49 15 43 28 2 36 59 Total Phenolic Content (mg GAE/g) 2014 Variety Number Figure 5 .b. Bound P henolic C ontents of 2014 MSU Wheat Breeding varieties. Variety numbers and corresponding names are listed in Table 1. Error bars represent one standard deviation of Difference test (p<0.05) a nd fully presented in Appendix C , Table 13 . Bound phenolic content is obtained by subtracting the free from the total phenolic contents. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 51 58 17 47 31 30 41 21 40 45 37 62 27 8 9 33 13 46 61 52 Bound Phenolic Content (mg GAE/g) 2014 Variety Number 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 49 34 12 60 14 5 24 42 39 6 15 38 29 2 23 36 28 59 43 Bound Phenolic Content (mg GAE/g) 2014 Variety Number Figure 5 .c. Free P henolic C ontents of 2014 MSU Wheat Breeding varieties. Error bars represent one standard deviation of measurements in triplicate. Significant groupings were analyzed with Least Significant Difference test (p<0.05) and fully presented in Appendix C, Table 13 . 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 43 62 38 6 23 17 12 24 27 29 30 61 42 40 5 28 34 31 9 Free Phenolic Content (mg GAE/g) 2014 Variety Number 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 59 39 14 47 48 60 33 15 13 36 52 2 37 8 51 58 46 21 41 49 Free Phenolic Content (mg GAE/g) 2014 Variety Number 4.2.2 . Total Phenolic Content s of Milling Fractions of Wheat Varieties For the TPC analysis of the 2013 and 2014 milled wheat fractions, the bran fractions consistentl y had the highest phenolic content values, followed closely by the shorts, with flour containing the lowest concentrations. When the TPC values for each studied fraction of the 34 repeated wheat varieties were averaged, the average TPC value of all shorts samples was approximately 80% of the average for all bran, and the average TPC value for all flour samples was approximately 20% of the average for all bran (Figure s 6 and 7 ). For the bran fractions, the highest measured TPC values were for varieties F2040 (#22) and F2011 (#32) fro m the 2013 harvest (Figure 6 ), and F2020 (#38) and Jupiter (#52) from the 2014 harvest ( Figure 7 ). The highest measured TPC values from the shorts fraction were for F2034 (#15) and F2011 (#32) from the 2013 harvest, and F1029 (#5) and F2039 (#24) from the 2014 harvest ( Figure 7 ). There were no clear patterns among the TPC values of the three milled fractions (i.e., the TPC levels did not correlate among fractions). The concentration of total phenolic compounds present in one fracti on did not seem to affect that present in other fractions. From the results of the wheat harvested in the two years, the ratio of phenolic contents present in the bran, shorts and flour fractions were approximately 5:4:1, res pectively. From these findings , bran and shorts should be considered the ideal starting materials for potential use for commercial phenolic acid extraction utilizing wheat milling byproducts. The above results also indicate that the potential removal of endosperm in future applications would be unlikely to cause any significant loss in phenolic contents due to the relatively lower amount of phenolic acids found in the white flour compared to bran . It needs to be pointed out that despite the lower concentrations of TP in flour fractions than in shorts and brans, white flour is the major constituent of the wheat milling streams (approximately 70 - 75%). So even though TP concentration in flour is only about 20% of that in bran, the milled white flour fraction does provide nearly half of the phenolic content of a whole milled wheat sample. The relatively lower concentrations of phenolic acids found in the refined white flour samples suggest that it might not be sufficient to provide meaningful health benefits from phenolic content upon the re moval of bran and shorts. This helps explain why whole grain wheat can provide more phenolic compounds, and help promote the physiological benefits of wheat. Figure 6 . Total P henolic C ontents of M illed Wheat F ractions from S elected Michigan wheat vari eties harvested in 2013. Samples were analyzed in triplicate and results are reported as mean values with one standard deviation. 0.0 2.0 4.0 6.0 8.0 10.0 12.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Total Phenolics Content (mg GAE/g fraction) 2013 Variety Number shorts bran flour 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Total Phenolics Content (mg GAE/g fraction) 2013 Variety Number shorts bran flour Figure 7 . Total P henolic C ontents of M illed Wheat F ractions from S elected Michigan W heat V arieties harvested in 2014. Samples were analyzed in triplicate and reported as mean values with one standard deviation. 0 2 4 6 8 10 12 14 2 5 6 8 9 12 13 14 15 17 21 23 24 27 28 29 30 31 33 34 Total Phenolic Contents (mg GAE/g fraction) 2014 Variety Number shorts bran flour 0 2 4 6 8 10 12 14 16 36 37 38 39 40 41 42 43 45 46 47 49 51 52 58 59 60 61 62 Total Phenolic Contents (mg GAE/g fraction) 2014 Variety Number shorts bran flour 4.3 . Ferulic Acid Content of Whole Ground Wheat Flour Alongside the MSU wheat breeding varieties, three commercial varieties, Aubrey, Ambassador, and Jupiter, we re also harvested, milled to whole ground flour , and quantified for ferulic acid contents ferulic acids, i.e., bound and free, with bound ferulic acid being the majority. The mean value s of the free ferulic acid contents composed only 5% of that of the total ferulic acid for both 2013 and 2014 harvest year varieties (Tables 4 and 5 ). There did not appear to be any correlation between the amount of bound and free ferulic acids, presumably because the amount of free ferulic acid was too minuscule to indicate any specific pattern. From results in the present study of selected Michigan wheat breeding varieties , the bound (i.e., water insoluble) form of ferulic acid was the only significant ly large component of the phenolic compounds found. Table 4 . Ferulic Acid Contents 1 of Selected 2013 MSU Wheat Breeding Varieties Variety Number Variety Name Total FA Content ( g FA/g) Bound FA Content ( g FA/g) Free FA Content ( g FA/g) 1 F1014 362 ±6 351 ±11 11.4 ± 5.4 2 F1026R 302 ±6 290 ±8 12.5 ± 1.8 3 F1012 310 ±24 297 ±17 13.5 ± 7.1 4 F1003R 250 ±9 234 ±6 16.4 ± 3.7 5 F1029 326 ±7 314 ±9 12.4 ± 2.3 6 F1047 288 ±16 274 ±11 14.4 ± 5.6 7 F1032R 309 ±3 298 ±2 11.4 ± 1.1 8 F1027 350 ±2 336 ±2 13.6 ± 0.4 9 F1049 309 ±9 298 ±12 11.0 ± 2.9 10 F1051 261 ±1 240 ±1 21.5 ± 0.4 11 F1050 358 ±13 328 ±17 30.1 ± 3.7 12 F1048 337 ±27 314 ±35 23.5 ± 7.9 13 F2031 313 ±3 299 ±4 13.6 ± 1.2 14 F2033 272 ±3 246 ±2 25.7 ± 1.2 15 F2034 293 ±1 282 ±1 11.5 ± 0.4 16 F2032 373 ±22 357 ±2 15.6 ± 6.0 17 F2037 314 ±2 294 ±1 19.6 ± 0.6 18 F2035 360 ±33 348 ±29 12.5 ± 11.9 19 F2041 341 ±13 320 ±9 21.5 ± 3.8 20 F2036 253 ±11 240 ±16 12.6 ± 4.5 21 F2038 297 ±2 285 ±1 11.6 ± 0.7 22 F2040 257 ±12 238 ± 17 19.5 ± 4.5 23 F2042 371 ±14 345 ±18 25.6 ± 4.1 24 F2039 412 ±1 399 ±1 12.7 ± 0.4 25 F2001R 326 ±30 308 ±20 18.5 ± 10.0 26 F2006 235 ±12 216 ±17 19.4 ± 5.2 27 F2005 318 ±3 293 ±4 25.2 ± 1.0 28 F2015 370 ±12 355 ±16 15.4 ± 3.2 29 F2009 321 ±7 304 ±5 17.5 ± 2.4 30 F2012 285 ±30 272 ±44 12.9 ± 14.3 31 F2008 339 ±4 312 ±5 27.5 ± 1.3 32 F2011 365 ±26 348 ±13 17.4 ± 7.0 33 F2014R 283 ±12 267 ±8 16.4 ± 4.2 34 F2003 390 ±5 374 ±4 15.8 ± 1.3 35 F2004 355 ±19 332 ±24 23.5 ± 5.3 36 F2002 318 ±8 302 ±10 15.9 ± 2.5 37 F2028R 325 ±10 306 ±13 18.7 ± 3.1 38 F2020 293 ±22 285 ±14 8.5 ± 7.6 39 F2024R 265 ±1 249 ±1 15.6 ± 0.5 40 F2018 365 ±10 339 ±13 26.4 ± 2.7 41 F2019 285 ±15 274 ±19 11.5 ± 5.2 42 F2016 289 ±26 274 ±15 14.6 ± 8.9 43 F2022 397 ±11 383 ±14 13.7 ± 2.6 44 F2025R 290 ±1 272 ±1 18.5 ± 0.2 45 F2021 310 ±40 290 ±34 20.3 ± 12.9 46 F2030 303 ±22 282 ±35 21.5 ± 7.1 47 F2029R 343 ±21 316 ±15 26.8 ± 6.1 48 F2027R 339 ±3 320 ±2 19.5 ± 1.0 49 Aubrey 312 ±12 297 ±16 14.7 ± 4.0 50 Hopewell 357 ±25 340 ±33 17.3 ± 6.9 51 Ambassador 357 ±12 338 ±16 19.4 ± 3.8 52 Jupiter 337 ±34 322 ±24 14.7 ± 10.1 53 Red Ruby 329 ±15 304 ±21 25.3 ± 4.6 54 VA09W - 188WS 272 ±12 257 ±17 14.6 ± 4.3 55 Cayuga 334 ±13 317 ±17 17.4 ± 3.9 56 VA09W - 192WS 265 ±19 251 ±12 13.7 ± 7.0 57 Caledonia 325 ±9 312 ±12 12.6 ± 2.8 1 Results are expressed as micrograms of ferulic acid standard equivalent per gram of whole ground flour, with the data presented as means ± standard deviation (n=2). For statistically significant groupings of data see Appendix C, Table 13 . Table 5 . Ferulic Acid Contents 1 of Select 2014 MSU Wheat Breeding Varieties Variety Number Variety Name Total FA Content ( g FA/g) Bound FA Content ( g FA/g) Free FA Content ( g FA/g) 2 F1026R 388 ± 4 370 ± 7 18.2 ± 3.4 5 F1029 419 ± 49 399 ± 49 19.3 ± 0.8 6 F1047 387 ± 39 351 ± 61 36.0 ± 7.8 8 F1027 375 ± 13 353 ± 1 22.4 ± 11.4 9 F1049 373 ± 2 356 ± 1 17.6 ± 0.8 12 F1048 403 ± 10 388 ± 10 15.1 ± 0.2 13 F2031 385 ± 20 363 ± 23 21.9 ± 3.1 14 F2033 338 ± 11 307 ± 7 31.4 ± 4.0 15 F2034 397 ± 20 373 ± 29 24.0 ± 8.6 17 F2037 370 ± 3 353 ± 3 17.3 ± 0.1 21 F2038 413 ± 9 399 ± 14 14.3 ± 5.2 23 F2042 346 ± 15 325 ± 11 21.1 ± 4.0 24 F2039 457 ± 41 429 ± 22 28.0 ± 18.7 27 F2005 318 ± 1 304 ± 0 14.1 ± 1.2 28 F2015 356 ± 7 341 ± 6 15.4 ± 1.2 29 F2009 403 ± 19 387 ± 19 16.1 ± 0.5 30 F2012 429 ± 0 415 ± 1 14.6 ± 0.7 31 F2008 390 ± 15 372 ± 16 18.3 ± 1.6 33 F2014R 457 ±2 4 435 ±3 4 22.9 ± 0.5 34 F2003 432 ± 35 415 ± 45 17.5 ± 0.3 36 F2002 368 ± 20 343 ± 25 25.1 ± 15. 5 37 F2028R 467 ± 34 442 ±4 1 25.0 ± 7.3 38 F2020 373 ± 2 356 ± 3 16.4 ± 4.8 39 F2024R 382 ± 27 363 ± 26 18.2 ± 1.3 40 F2018 368 ± 36 344 ± 39 23.2 ± 3.5 41 F2019 353 ± 30 329 ± 31 24.2 ± 1.1 42 F2016 396 ± 10 375 ± 9 20.7 ± 0.4 43 F2022 360 ± 51 341 ± 43 19.4 ± 2.2 45 F2021 381 ± 21 349 ± 27 32.9 ± 6.2 46 F2030 421 ± 28 390 ± 43 31.2 ± 15.0 47 F2029R 452 ± 26 429 ± 29 22.5 ± 2.5 49 Aubrey 334 ± 6 303 ± 23 30.9 ± 17.0 51 Ambassador 324 ± 1 305 ± 3 18.8 ± 2.0 52 Jupiter 316 ± 36 298 ± 34 18.6 ± 2.3 58 MSU Line F2010 371 ± 9 351 ± 21 20.0 ± 12.0 59 Unnamed 1 391 ± 36 374 ± 32 16.9 ± 3.2 60 Unnamed 2 366 ± 1 347 ± 6 18.7 ± 5.6 61 Unnamed 3 422 ±4 2 407 ± 44 15.9 ± 2.2 62 F0013R 368 ± 38 330 ± 33 37.8 ± 4.7 1 Results are expressed as micrograms of ferulic acid standard equivalent per gram of whole ground flour, with the data presented as means ± standard deviation ( n=2 ). For statistically significant groupings of data see Appendix C, Table 13 . The range of the total ferulic acid contents was between 235 g FA/g and 412 g FA/g for 2013 varieties (Table 4 ), with a 57% difference between the maximum and minimum measured values. The range of total ferulic acid contents was between 316 g FA/g and 467 g FA/g for 2014 varieties ( Table 5 ), with a 68% difference between the most extreme values. Among the 2013 w heat varieties, the highest values were for varieties F2039 (#24) and F2022 (#43), while the lowest values were for F2036 (#20) and F2006 (#26) ( Table 4 ). For the 2014 wheats, varieties with the highest measured total ferulic acid contents were F2039 (#24) , F2028R (#37), and F2029R (#47), while those with the lowest were F2005 (#27), Ambassador (# 51), and Jupiter (#52) ( Table 5 ). These ferulic acid content ranges were found to be consistent with the findings reported by Adom and colleagues (Adom et al., 2 003), and fall within their measured range of between 250 g/g and 513 g/g for durum and soft wheat varieties harvested from the Eastern United States. Mpofu and colleagues reported similar results for hard wheat varieties from Western Canada, at between 2006). Moore and colleagues (2005) measured soft wheat varieties grown in Maryland, USA, and their total ferulic acid contents range d between 455.5 g/g and 621 g/g, which was a higher range than many of the values mea sured in the current study , even though all of the studied Michigan varieties were soft wheats as well . The commercial varieties were all among the wheat varieties with lower measured ferulic acid contents for both crop years (Tables 4 and 5 ). In the 201 3 harvest, Aubrey had a total ferulic acid content of 312 g/g o f whole ground flour , Ambassador had a total ferulic acid content of 358 g/g, and Jupiter a content of 338 g/g. In the 2014 harvest, Aubrey had a total ferulic acid content of 334 g/g, Ambassador had a total ferulic acid content of 324 g/g, and Jupiter one of 316 g/g. On average, Aubrey had a slight increase in ferulic acid content in 2014, while Ambassador and Jupiter showed slight decreases. In other words, a vast majority of th e breeding varieties studied showed higher ferulic acid contents than some commercially grown soft winter wheat varieties in Michigan. This information may be helpful for breeders to identify potential early lines with higher ferulic acid and total phenoli c contents in their breeding programs. As expected, the ferulic acid (FA) contents of the varietie s were different from each other in their measured values. The total phenolic contents were higher than the total ferulic acid contents , due to minor contrib utions from other phenolic compounds. The FA levels had their highest and lowest values with different varieties. The commercial variety Ambassador (variety # 51) was among the lower FA values, while the values for Aubrey and Jupiter (varieties #49 and #52 ) were close to the median value ( Table s 4 and 5 ). Out of the three commercial varieties, Ambassador was consistent in having among the lowest values for both ferulic acid content and total phenolic content out of the 2014 studied varieties. 4. 4. Pearson Correlations of the Phenolic and Ferulic Acid Contents of Studied Varieties For further examination of the results of TPC by colorimetric analysis, the Pearson product - moment correlation coefficient was calculated between total and bound phenolic contents of the combined two harvest seasons using Microsoft Excel; this coefficient was 0.97 (n=34), indicating a strong positive relationship. This confirms that the TPC was mainly impacted by the bound PC. On the other hand, the correlation coefficient between the total phenolic contents and the respective free phenolic contents for the whole ground flour of the studied varieties was - 0.1, which demonstrated no correlation. These results show that the amount of free phenolic compounds could not indicate the amou nt of bound phenolic compounds present for a given variety. In fact, whether a variety contained relatively high or low amounts of bound phenolic acids, the free phenolic content remained negligible , ranging between about 5 and 10% . If the bound phenolic a cids in the flour samples were actually being released into free form through enzymatic or chemical hydrolysis over time, a pattern of ratios between the bound and free phenolic content s could not be found in this study to support this hypothesis. The free phenolic contents of most varieties studie d were close to 2.0 mg GAE/g, whereas the values of the bound phenolic contents were much more varied and had larger ranges. For the whole ground wheat flour samples , the Pearson product - moment correlation coefficient between the total phenolic contents measured by the Folin - Ciocalteu assay and the total ferulic acid contents measured by RP - HPLC was 0.26 (n=35), which indicated a weak positive relation between the two sets of data. Most varieties with relatively high total phenolic content were also relatively high in ferulic acid content, but the varieties with the highest or lowest recorded values were not the same for the two methods studied. The main reason for the discrepancies in results bet ween the two methods could be the specificity for and range of the compounds that were measured and identified. The Folin - Ciocalteu assay measures the entire reducing capacity of the sample, mainly phenolic compounds in plant materials such as wheat. That means all phenolic compounds will contribute to the results, and any kind of non - phenolic compounds may react and influence the readings. Such compounds could be other types of antioxidants present in wheat, such as carotenoids and , proteins, thiols and fr ee sugars , and tocopherols (Moore et al., 2005) . For some of the wheat varieties examined in the current study , clear differences in flour suspension color brightness were noticed during alkaline hydrolysis of bound phenolic contents, which suggested that the varieties could have contained different types of non - phenolic antioxidants which reacted differently when exposed to the alkaline pH environment. This discrepancy between actual measured substances may help explain the poor correlation between total ferulic acid and total phenolic content among the wheat varieties studied. Overall, the studied Michigan wheat varieties had phenolic acid contents similar to those measured and reported elsewhere in the United States (Adom et al., 2003) . High and low phenolic acid ranges were found, although, the difference s were not extreme. However, that did not dismiss the possibility of the existence of genetic far outliers that may still exist within the Michigan wheat breeding varieties. 4.5 . Effect of Crop Yea r on Ferulic Acid Content of Whole Ground Wheat Flour Samples Of the studied wheat varieties, a total of 34 were planted and harvested in both 2013 and 2014 in the central Michigan region of Saginaw and Ingham. They were compared for annual changes in ferulic acid contents. Out of 34 comparable varieties, 29 varieties showed increased total ferulic acid (FA) content in the 2014 harvest when compared to the 2013 harvest, with 14 varieties having increases greater than 30%, and 4 varieties showing small decreases of up to 10%. The average total FA content of the 34 comparable varieties harvested in the two crop years s howed an increase of 23% from 2013 to 2014, from 314 g FA/g whole ground flour to 386 g FA/g, respectively. From one - way ANOVA analysis, the measured total FA values of the 2013 and 2014 crops studied were statistically significant ly differen t (p<0.001). It is well known that plant traits are generally governed by genetic and environmental factors, and it is not surprising that the crop year, as an external factor, asserted significant impact on the phenolic contents (i.e., FA in the current study) of the wheat kernels. Since these wheat varieties were planted in the same experimental location with controlled fertilizer applications, the weather conditions became the most important variations for consideration. Soil and weather conditions for the planting sites of the east - central region of Michigan were acquired from the Michigan State University Enviro - weather Automated Weather Station Network (MAWN; http://www. agweather.geo.msu.edu.proxy1.cl.msu.edu/mawn/mawn.html ), between the months of September from the previous year, at planting, to June of the harvest year ( select data listed in Table 6 ; for all data, please see the website data listed for east - central Mich igan from September 1, 2012 through June 31, 2013, and from September 1, 2013 through June 31, 2014). In the crop year of 2013, the highest recorded daily air temperature for 24 hours was 14.7 o C to 33.1 o C, while the lowest recorded single day air temperatu re range was - 11.7 o C to - 7.8 o C. For the growing seasons of crop year 2014, the highest recorded daily air temperature range was 10.9 o C to 30.9 o C, while the lowest recorded 24 - hour range was - 25.3 o C to - 17.0 o C. From these data, the temperature range showed a much colder winter in 2014. The measured cumulative rainfall was 534 mm in the 2013 crop growing period, and dropped by 55% to 242 mm for the 2014 crop growing period. In the 2013 planting season, the highest measured daily soil moisture at 10.16 cm dep th was 0.947 - 1.082 cm water /cm soil , and the lowest daily soil moisture was 0.274 - 0.277 cm water /cm soil . In the 2014 planting, the highest soil moisture measured at 10.16 cm depth was 0.980 - 0.986 cm water /cm soil , while the lowest soil moisture was 0.345 - 0.345 cm water /cm soil . The average soil moisture had increased from 0.546 cm water /cm soil in 2013 to 0.622 cm water /cm soil in 2014. There are many environmental factors that can cause changes in ferulic acid content in many plant species, such as high irradiation, heat or chilling, excessive change in moisture caused by droughts or flooding, and nutrient deficiency (Schützendübe and Polle, 2002). For the soil moisture, a decrease in soil moisture could decrease the nutrient absorption of crops (Metwally and Pollard, 1959), and the subsequent mineral deficiency could signal increased production of phenolic acids in laboratory situations (Marschner, 1991 ). Both the lowest measured soil moisture and the average soil moisture had increased from 2013 to 2014 . Thus, soil moisture was probably not a main factor behind the changes in the total ferulic acid of the studied wheat varieties of two crop years. Decreased rainfall could lower the overall water content in the crops and cause drought - and saline - induced stress responses, but this can be compensated for by increased watering and/or irrigation. The lack of decrease in soil moisture showed that the decrease in rainfall did not seem to be a factor. Moreover, Mpofu and colleagues found no correlation between t he average rainfall and the phenolic contents of different wheat varieties grown in Western Canada (Mpofu et al., 2006). Members of the grass family increase the production of phenolic compounds to counter cold - induced oxidative stress (Sarkar et al., 200 9). Decreases in air temperature have also been known to increase the presence of phenolic compounds in non - cereal plants (Rivero et al., 2001). Michigan had experienced an unusually cold winter in the winter prior to harvest season of 2014. Since the othe r soil and weather factors have been eliminated as major contributors, it is possible that the low average daily air temperatures experienced for the 2014 crop from October 2013 to March 2014 had a direct effect on the ferulic acid content of the studied M ichigan wheat varieties. This indicates that cr ops adapted to colder climates, such as winter wheat, may possess superior genetic capacity for increased phenolic acid biosynthesis compared to crops grown in warmer climates, as part of their adaptation stra tegy. Table 6 . Select Environmental conditions of Growing Locations in East - Central Michigan from the H a rvesting and P lanting S easons of 2013 to 2014 (MAWN, 2012, 2013, 2014) Year of Harvest Maximum measured single day average air temperature ( o C) Minimum measured single day average air temperature ( o C) Total Rainfall (cm) Minimum Measured Soil moisture (cm water /cm soil ) Average Daily Soil Moisture (cm water /cm soil ) Average Crop Total Ferulic Acid content ( g FA/g) 1 2013 33.1 - 9.75 53.42 0.277 0.546 290 2014 30.9 - 21.15 24.23 0.345 0.622 316 1 Average of 34 wheat varieties studied; g FA/g whole ground flour. 4. 6 . Baking Quality of Cookies after Fortification with Free and Bound Forms of Ferulic Acid A model system of white flour for cookie baking was used to help understand the stability of free and bound forms of ferulic acid during simple processing like cookie baking. Ambassador was used as the representative variety for the cookie baking trials . A ll cookie treatments were baked in triplicate. The cookie moisture content, cookie color, dimensions, and hardness were examined after baking to compare the baking qualities of the different flour blends with and without added ferulic acid. 4.6 .1 Visual Quality of Cookies Baked from Flour Fortified with Free and Bound Forms of Ferulic Acid Cookies made from the treatment flour blends with the 20% bran and the free FA powder most resembled the control white flour cookie visually by possessing similar distr ibution of brown spots and surface ridging (Figure s 8 . b and 8 . d , respectively ). Cookies from the treatment using the 4.4% arabinoxylan blend were noticeab ly smoother in texture (Figure 8 . c). The treatment with the free FA powder produced cookies with sligh t edge darkening (Figure 8 . d), while cookies made from the flour blend with the digested 20% bran and free FA powder showed sign i ficant edge darkening (Figure 8 . e), with noticeable dark brown and black spots spread throughout its surface. Figure 8 . R epresentative Visual Appearance of Cookies b aked using D ifferent F lour Blends C onsisting of white flour and: (a) N o additions, (b) 20% br an, (c) 4.4% arabinoxylan, (d) F ree FA powder, and (e) D igested 20% bran with free FA powder. a b c d e 4.6 .2. Effect of Ferulic Acid Source on Cookie Moisture Contents Ambassador flour was used as the representative variety. The highest cookie moisture was observed for cookies baked from the flour blend with 4.4% wheat arabinoxylan as a source of ferulic acid fortification (Table 7 ). The moistures of the baked cookies made from the 4.4% arabinoxylan flour blend and the 20% bran flour blend were significantly higher than the moisture contents o f the baked cookies from the control and the other flour blends. In addition, there were no significant differences in baked cookie moisture among cookies made from the control flour and the flour blends with free FA or with 20% digested bran and free FA (Table 7 ). There was strong positive Pearson correlation of 0.98 between the combined final cookie moisture content and the cookie height ( A ppendix A , Table 11 ). It is believed that the arabinoxylan, i.e., the 20% bran flour blend) or insoluble arabinoxylan isolated from wheat bran (i.e., the 4 .4 % arabinoxylan flour blend) resulted in markedly higher cookie moistures because of the high water absorption capability of arabinoxylan. Presen ce of t he bran that had been treated with NaOH hydrolysis to remove bound ferulic acid then supplemented with FFA (i.e., the 20% digested bran plus free FA flour blend) did not seem to have a large impact on water absorption and final moisture content of t he cookies. It is possible that the alkaline treatment and heating had decreased the water absorption capability of arabinoxylan. Table 7 . Moisture Contents 1 of Cookies Baked From Different Ambassador Wheat Flour Blends 1 Values with the same letter are not 6 .3. Effect of Ferulic Acid Source on Cookie C olor Color analysis was performed on the different flour blends from the Ambassador flour. The color and lightness of cookie samples were measured using the LAB Colorspace scale, corresponding to the value L, where 0 is the darkest and 100 is the lightest. The opposing color axis was measured using a* and b*, which showed the cookies as all within the brown color region . The lightest baked c ookies were from the flour blend fortified with wheat arabinoxylan powder at 73.2 lightness (L) units, and from the 20% bran flour blend at 70.4 lightness units (Table 8 ). The cookies with the lowest LAB values were from the flour blend with 20% digested b ran flour fortified with free FA powder, which was significantly different from all other samples. The LAB values of cookies baked from each of the treatment flours were also significantly different from that of cookies baked from the control white flour. The cookies with the lightness values closest to that of the control cookie were those made from the 20% bran flour blend, which was 3 units lighter, and from the white flour fortified with FA powder, which was 3 units darker. For the cookie color axis (a* and b*), all of the flour blend cookies, except those made from the free FA blend, were significantly different from the control in color. In cookie baking, the main factor in changes to cookie color is due to non - enzymatic browning. This process is mois ture sensitive, as the increased presence of even small amounts of water in foods during cooking can drastically reduce the degree of Maillard reaction (Peterson et al., 1994). This could explain the significantly lighter color of the bran cookies and the arabinoxylan cookies in the present study, as both of these cookie samples also had significantly higher moisture contents (Table 7 ). Table 8 . Color A nalysis 1 of F erulic A cid - Fortified C ookies made from Amba ssador flour 1 Values not sharing the same letter in the same column are significantly different from each other (p<0.05). L* indicate s lightness, a* represents the red/green color axis, and b* represents the yellow/blue axis. Non - starch po lysaccharides have been known to pull water away from the flour (James et al., 1989), but the bran and arabinoxylan cookies did not show increased browning even though white flour was still the majority ingredient for all the flour blends . Therefore , the amount of water held by the non - digested bran and arabinoxylan may have been either redistributed to the white flour later on during baking, or the quantity of water added in the cookie formulation was already above the absorption limit of the white f lour portion, with the unabsorbed water taken up into the bran and arabinoxylan polysaccharides. The browning of the digested bran blend cookies was very extreme, being much higher than the slight browning of the FA powder blend cookies, despite being sim ilar in moisture content relative to the cookies baked from the FA flour blend. This indicates that one or more enzymatic reaction s had possibly taken place. Polyphenol oxidase is a common browning enzyme in plants and is present in many wheat varieties (D emeke and Morris , 2002). In the literature, enzymatic browning has been reported by others in wheat - based food products such as noodles (Fuerst et al., 2006). From studies performed by Yang and colleagues, polyphenol oxidase was stable at pH 5 - 11, and temp eratures of up to 70 o C (Yang et al., 2000). The temperature and pH conditions of the bran alkaline digestion and drying process in Section 3.2.7 were well within the ranges to permit retention of polyphenol oxidase in the digested bran , allowing for a small time window for enzymatic browning . T he non - digested bran must have contained identical amounts of the same browning enzymes, but they were not able to react with the bound ferulic acid to produce browning. It is possible that the undigested bran cel l wall may have remained structurally stable for sufficient time to prevent the release of intracellular enzymes until their heat inactivation, preventing their reaction with other cookie ingredients, thereby limiting the a mount of browning that occurred. Ferulic acid begins to decompose at 203 o C (Fiddler et al., 1967), and the cookie baking temperature of this experiment was 204 o C. The breakdown of ferulic acid may have released its phenolic components , which facilitated reaction with PPO. It was also obse rved in the present stu dy that the color lightness (L*) and redness (a*) appeared to be related to the form of ferulic acid, free vs. bound. Compared to the values for the control sample of white flour cookies, the addition of free FA (samples #4 and #5) i ncreased both darkness and redness values, while the cookies with added bound FA at the same levels (samples #2 and #3) had reduced darkness and redness . Besides the impact of water holding capacity of arabinoxylan (James et al., 1989) and higher cookie mo isture discussed above, the form of FA may play a role in affecting the Maillard reaction, with bound FA being less available to react with the other flour ingredients, as evidenced by the cookies baked with the non - digested bran flour blend. Using the Pea rson correlation equation, there was a weak positive correlation of 0.38 between the decrease in total cookie ferulic acid content and decrease in cookie color lightness (Appendix A, Table 11 ). 4.6 . 4 . Cookie D iameter and H eight From the cookie dimension measurements, cookies baked from flour blends with the arabinoxylan and with the bran had statistically significantly greater cookie heights at 1.3 cm and 1.2 cm, respectively, than the other three cookie samples , which had simila r lower heights that were not significantly di fferent from each other (Table 9 ). The cookie diameters of all the blends and control were significantly different from each other, and the diameters of the cookies baked from the 20% bran blend and 4.4% arabin oxylan blend were both less than the diameter of the cookies from the control white flour ( Table 9 ). Polysaccharides such as cellulose and arabinoxylan are more rigid than amylose and amylopectin, and have been known to cause decrease in spread when added to cookie blends (James et al., 1989) . Moreover, increased cookie moisture can decrease the glass transition temperature, which lowers cookie spread (Miller et al., 1997) , and the presence of polysaccharides was associated with substantially increased coo kie moisture in the current stu dy (Table 7 ) . Table 9 . Physical Q ualit ies 1 of C ookies B aked from Different B lends of Ambassador Wheat Flour F ortified with Identical A mounts of Ferulic A cid (n=3) 1 Values that do not share the same letter in the same column are significantly different from each other (p<0.05). Hardne ss was measured by the peak force of cookie breakage. 4.6 . 5 . Cookie H ardness The control cookies were the softest with 22 N peak force requ ired to break the cookies , while the flour blend cookies with 4.4% arabinoxylan and with FA powder produced the hardest cookies, with both having 29 N peak force values (Table 9 ). The digested and non - digested bran cookies were each harder than cookies of the control white flour, but softer than those of the arabinoxylan and FA powder blends. Differences in baked product moisture did not significantly impact the hardness of the cookies, so case hardening from internal moi sture reloc ation could not have been the likely cause of the difference in cookie hardne ss . The intermediate cookie hardne ss baked from flour blends with 20% dige sted (#2) or undigested bran could be due to the decreased starch content and increased cellulose content of those blends, which could have increa sed dough compactne ss (Gujral et al., 2003). The arabinoxylan blend cookies had high moisture content, yet the greatest hardness value, which indicates that water retention did not soften the cookies. This is in con firmation with other reports of cookies fortified with arabinoxylan oligosaccharides showing increased baked cookie hardness (Pareyt et al., 2011). 4.6 . 6 . Changes in T otal F erulic A cid C ontents after Baking The total ferulic acid (FA) contents of all the cookie samples (in ground powder form) were measured using RP - HPLC as described in Chapter 3. The procedure used was the same as in the digestion of whole ground flour, as ground cookie powders had well dispe rsed in the alkaline solution without complications. From the results obtained in this study, there were noticeable losses in the FA contents of the baked cookies made from all of the flour blends , including the losses in the naturally occurring bound feru lic acid in the undigested bran and arabinoxylan blends ( Figure 9 ). The blends (#2 through #5) all u sed flour milled from the Ambassador wheat variety. They were all formulated to the same FA content of 324 g/g, which wa s based on the FA content of the g round whole ground flour from Ambassador. The undige sted bran and arabinoxylan contained naturally occurring bound FA, and were added to the white flour proportionally so that the total FA content of the flour blend sample reached the same amount as that of ground whole ground flour from Ambassador. For all blends (except control) , t his translated to 180 g FA/g of mixed cookie dough (Figure 9 ) . The 20% bran flour blend had the highest FA content remaining after baking, at 155 g FA/g ground baked cookie p owder . This was a drop from the initial 180 g FA/g , for a 90% retention rate. Cookies made from t he flour blended with 20% digested bran fortified with free FA had the lowest amount of FA remaining, with 38% FA retention (Figure s 9 and 10 ). The amount o f FA remaining in the baked cookies made with the 4.4% arabinoxylan blend (sample #3) and the free FA blend (sample #4) were very similar, at 98 (54%) and 95 (53%) g FA/g ground baked cookie powder, respectively. From the analysis of the data, there was n o significant difference in the post - baking FA retention between samples #3 and #4 , showing evidence of considerable amount of ferulic acid oxidation in both cookie blends from the massive decrease in measured ferulic acid content . The loss of ferulic acids during baking of the digested bran blend (sample #5 ) indicated that there was more than a physical mechanism due to differences with the undigested bran sample , and it is highly likely that the polymerization of lignin - carbohydrate cross - link ed complexes had a critical role in stabilizing ferulic acid by the incorporation into the lignin - network within the plant cell wall (Ralph et al., 1995) , which may have been the cause for the preservation of the bound ferulic acid in the undigested bran c ookie after baking (20% bran blend cookies vs. 20% digested bran with free FA blend cookies) . The lack of similar FA preservation in the arabinoxylan blend despite also being in the bound form gives further evidence to the important role played by bran. The free FA blend cookies showed identical amounts of FA loss as the arabinoxylan blend cookies, which will be elaborated further in Section 4.6.6. The similar light coloring of the cookies baked from both of these blends showed that the lost FA did not pa rtake in browning reactions, which provides evidence of possible ferulic acid cross linkage with flour proteins. When ferulic acid is oxidized into quinones, it can chemically bond with the amino and thiol groups in proteins (Figueroa - Espinoza et al., 1999 ). The free radical of ferulic acid can also react with the tyrosine groups of proteins to form diferulic acid, which is a crosslinking agent for polysaccharide chains (Vansteenkiste et al., 2004). The addition of ferulic acid to flour in quantities simila r to this study has been known to decrease gluten crosslinking and gluten elasticity (Koh and Ng, 2009), which would result in increased hardness and brittleness in low moisture baked products. The amount of ferulic acid remaining after baking had the high est Pearson correlation with cookie hardness out of all other cookie properties at 0.77 (Appendix A, Table 11 ). Based on the results of this study, the addition of undigested natural bran to flour would be the most cost effective approach of ferulic acid f ortification in the perspective of maximizing ferulic acid retention, as it requires the least chemical and physical processing other than size reduction of the bran particles. However, baking using digested bran has shown that the breakdown of bran struct ure aided in the degradation of ferulic acid and led to possible enzymatic browning of cookies . This means that any potential industrial application using wheat bran flour blends need to take bran stability and storage into consideration . The addition of arabinoxylan powder had shown no advantages over using free ferulic acid powder in decreasing ferulic acid loss during baking, and wa s the least cost - effective approach among the four different flour blends studied. With the most direct and cheapest fortification method by the ad dition of bran being the most effective, the fortification of ferulic acid in flour - based food products is absolutely technically feasible and economically viable, as long as the products are able to utilize whole gr ound flour and/or wheat bran. The proportion of bran added to the cookies was a moderate amount , meaning that the data of this study is directly applicable to commercial whole ground flour. F or simplicity of the model, the shorts fraction was not added to the blend for testing in order to avoid confusion due to differences in the composition between bran and shorts. Figure 9 . Total Ferulic Acid C ontent of B aked C ookie S amples made from V arious White F lour B lends. Bars not sharing the same letter are significantly different from each other (p<0.05). The blue line is an indication of the initially fortified level mea sured in the cookie dough of the treatments before baking, excluding the original control flour . The f erulic acid content of the control cookie without fortification wa s included for reference. a b c c d 0 20 40 60 80 100 120 140 160 180 white flour (control) 20% bran 4.4% arabinoxylan free ferulic acid 20% digested bran + free ferulic acid Total ferulic acid content ( g FA/g cookie) Flour Blend . Figure 1 0 . Percent of Ferulic A cid R etained in C ookies after B aking Cookie D oughs made from F our D ifferent W hite F lour B lends of W hea t Variety Ambassador . 0 10 20 30 40 50 60 70 80 90 100 20% bran 4.4% arabinoxylan free ferulic acid 20% digested bran + free ferulic acid Total ferulic acid remaining after baking (%) Flour blend CHAPTER 5 CONCLUSIONS AND FUTURE RECOMMENDATIONS During the first experiments of this thesis, the viability of enzymatic digestion versus alkaline digestion of ground whole ground wheat flour was compared, for the purpose of ferulic acid extraction a nd quantification. Although the enzymatic digestion method initially showed promise in extracting substantial amounts of ferulic acid, the sheer amount of enzymes required to reach the highest optimal extraction time made the procedure financially unfeasib le for use in experiments involving large numbers of sample materials. The time of the entire enzymatic digestion process was longer than the alkaline digestion method with a more complex sample preparation step. By taking these factors into consideration, the enzymatic digestion method was deemed inefficient for the scope of the experiments for the current study, and its potential use was discarded in favor of the alkaline digestion method for the extraction of total phenolic contents and ferulic acid cont ents from the ground wheat flour. After the total phenolic contents and ferulic acid contents of the selected Michigan wheat varieties were quantified, the results showed a general increase in detectable phenolic acids from wheat varieties of the 2014 h arvest compared with those of the 2013 harvest . In addition, the number of s ignificantly different variety groupings decreased in the 2014 harvest season. Using the data obtained from the Michigan Enviro - W eather P rogram, the most likely environmental factor causing the overall increase in phenolic acid synthesis in the crops was the severe cold winter of the 2014 season. In future experiments, growing wheat samples in controlled temperatures and deliberately exposing them to consistent lo wer air temperature may help in detecting changes in gene expression and identify the genes responsible for the increased phenolic acid synthesis. This could in turn assist in the breeding of potential new wheat varieties with the purpose of yielding incre ased or decreased phenolic acid contents. From the cookie baking experiments conducted with different cookie flour blends fortified with ferulic acid, the antioxidant retention was highest in the flour fortified with bran containing bound ferulic acid. T he cookies showing the least amount of changes in baking quality w ere made from the flour blend with added free ferulic acid powder. This type of cookie had the least amount of differences in physical appearances , such as color and dimension, but had signi ficantly the greatest losses in ferulic acid upon baking. Aside from the digested bran flour blend with released polyphenol oxidase , the addition of ferulic acid to the wheat flour generally increased baked cookie lightness. The water retention ability of the bran and arabinoxylan affected cookie hardness, spread and height ; ferulic acid oxidation - induced protein cross - linkage was also another possible factor in increasing cookie hardness. Of the four blends studied, t he bran flour blend was the most cost e ffective and had the highest ferulic acid retention levels after cookie baking . In future experiments, baking with modifications of the cookie formulation could be performed in order to minimize differences in cookie quality between the bran flour blend an d the control white flour blend. This could involve changes to the amount of any number of the wet and dry ingredients, such as flour, water, shortening, brown and white sugars, corn syrup, and ammonium bicarbonate. APPENDI CE S APPENDIX A . Supplementary Data for Wheat Phenolic Quantification and Cookie Baking Table 1 0 . Preparation of F erulic A cid S tandard S olution for RP - HPLC A nalysis Tube # Ferulic acid conc. (ppm) 100ppm Ferulic acid solution volume (ml) Methanol volume to pipette (ml) 1 10 0.5 4.5 2 20 1.0 4.0 3 30 1.5 3.5 4 40 2.0 3 5 50 2.5 2.5 6 60 3.0 2 7 70 3.5 1.5 8 80 4.0 1 9 90 4.5 .5 10 100 5.0 0 Table 11 . Pear s on Product - moment Correlation Coefficient between Different P arameters from Cookie B aking - - - - - Table 1 2 . Significant D ifferences of Total P opulations M easured us ing O ne way ANOVA with Fisher C omparison. Populations with p<0.05 were deemed significantly different Figure 1 1. UV - Visible S pectr um of Ferulic Acid Extracted from Michigan S oft Wheat Whole Ground Flour (Ambassador) . APPENDIX B. Viability of Enzymatic Digestion of Wheat Flour with Feruloyl Esterase Testing of the Ambassador variety flour with different enzyme combinations without a 30 - minute heat pre - treatment of the samples on a hot plate at 100 o C did not yield any significant f erulic acid release, except for small amounts of free phenolic acids (Appendix B , Figure 1 2). Flour samples that were given heat pre - treatment before the introduction of enzymes showed greatly increased ferulic acid (FA) measurements (Appendix B , Figure 1 3), indicating interaction of the added enzymes with the gelatinized starch constituents and the subsequent release of ferulic acid. Figure 1 2. Ferulic Content of Wheat Sa mples T reated with Constant Xylanase at 20 U and increasing amounts of Feruloyl Esterase (see Table 2, without pre - heating treatment). 1.04 1.14 1.24 1.34 1.44 1.54 1.64 1.74 1.84 1.94 0 5 10 15 20 25 30 35 Ferulic acid content (mg GAE/g) feruloyl e sterase enzyme activity unit (U) The increase in the amount of enzymes added to the flour samples corresponded to the release of ferulic acid ( Appendix B , Figure 1 4 ). The treatment with increasing levels of feruloyl estera se (FE) correlated with a gradual increase in measured FA while treatment with increasing levels of xylanase released higher amounts of phenolic acids at lower concentrations (Appendix B , Figure 1 3, 1 4). From results, the experiment showed that a proportio nally smaller amount of xylanase (5 U) was required for the optimal release of ferulic acid from the flour when compared to amount of FE required at 20 U ( Appendix B , Figure 1 3, 1 4 ). The TPC measurement of the representative variety Ambassador in the curr ent experiment was 3.1 mg/g Gallic Acid Equivalents (GAE). The Ambassador sample colorimetric measurement by alkaline extraction after defatting and washing with ethyl acetate was 3.5mg/g GAE. The proportion of the TPC from enzymatic digestion in compariso n to that from alkaline digestion is reasonable, as other phenolic acids were not extracted by the enzymatic method. Overall, the total time for sample preparation, digestion, and collection for enzymatic digestion was approximately 5 hours, compared to 4 hours for the alkaline digestion. However, the amount of enzymes required for optimal digestion was very high. This method was not cost effective for extracting ferulic acid from large numbers of different samples, and it was not economically feasible to employ this method for all of the wheat varieties for the rest of the experiments of this thesis. However, the enzymatic digestion method can still be useful for certain situations where isolation of ferulic acid and minimizing the presence of other starch constituents would be desired. Figure 1 3 . Ferulic Acid Content of Wheat S amples T reated with C onstant FE at 20 U and I ncreasing C oncentrations of Xyl (Table 2, with heating). 0 0.5 1 1.5 2 2.5 3 3.5 4 0 5 10 15 20 25 Ferulic acid contents (mg GAE/g) Xylana se activity unit (U) Figure 1 4 . Ferulic Acid Content of Wheat S amples Treated With Constant Levels of Xy l at 20 U , and Increasing Additions of FE , With Heat Pre - T reatment 1 . 1 For details, see Tab le 2 . 0 0.5 1 1.5 2 2.5 3 3.5 0 5 10 15 20 25 Ferulic acid contents (mg GAE/g) enzyme activity unit A PPENDIX C . Statistical Analysis Raw Data 2013 Factor Information Factor Levels Values C1 57 E0028, E1007R, E5011, F1003R, F1012, F1014, F1026R, F1027, F1029, F1032R, F1047, F1048, F1049, F1050, F1051, F2001R, F2002, F2003, F2004, F2005, F2006, F2008, F2009, F2011, F2012, F2014R, F2015, F2016, F2018, F2019, F2020, F2021, F2022, F2024R, F2025R, F2027R, F2028R, F2029R, F2030, F2031, F2032, F2033, F2034, F2035, F2036, F2037, F2038, F2039, F2040, F2041, F2042, I5440, I7067, I7127, I7826, I9339, I9340 2014 Factor Information Factor Levels Values C1 39 E0028, E5011, F0013R, F1026R, F1027, F1029, F1047, F1048, F1049, F2002, F2003, F2005, F2008, F2009, F2010, F2012, F2014R, F2015, F2016, F2018, F2019, F2020, F2021, F2022, F2024R, F2028R, F2029R, F2030, F2031, F2033, F2 034, F2037, F2038, F2039, F2042, I7826, x1, x2, x3 Table 13 .1. Raw Data for 2013 Total Ferulic Acid Content One - way ANOVA: C2 versus C1 Method Null hypothesis All means are equal Alternative hypothesis At least one mean is different Equal variances were assumed for the analysis. Analysis of Variance Source DF Adj SS Adj MS F - Value P - Value C1 56 0.17683 0.003158 10.95 0.000 Error 57 0.01643 0.000288 Total 11 3 0.19327 Model Summary S R - sq R - sq(adj) R - sq(pred) 0.0169801 91.50% 83.14% 65.99% Means C1 N Mean StDev 95% CI Ambassador 2 0.35762 0.01308 ( 0.33358, 0.38167) Aubrey 2 0 .31268 0.01227 ( 0.28864, 0.33672) Caledonia 2 0.32529 0.00906 ( 0.30124, 0.34933) Cayuga 2 0.33490 0.01274 ( 0.31086, 0.35894) Hopewell 2 0.3579 0.0247 ( 0.3339, 0.3819) Jupiter 2 0.3379 0.0337 ( 0.3139, 0.3620) MSU Line F1003R 2 0.24959 0.00931 ( 0.22555, 0.27363) MSU Line F1012 2 0.3103 0.0219 ( 0.2862, 0.3343) MSU Line F1014 2 0.36218 0.00562 ( 0.33813, 0.38622) MSU Line F1026R 2 0.30223 0.00551 ( 0. 27818, 0.32627) MSU Line F1027 2 0.35091 0.00156 ( 0.32687, 0.37495) MSU Line F1029 2 0.32584 0.00718 ( 0.30179, 0.34988) MSU Line F1032R 2 0.30975 0.00312 ( 0.28571, 0.33379) MSU Line F1047 2 0.2882 0.0162 ( 0.2642, 0.3 123) MSU Line F1048 2 0.3376 0.0268 ( 0.3136, 0.3617) MSU Line F1049 2 0.30909 0.00859 ( 0.28505, 0.33314) MSU Line F1050 2 0.35835 0.01303 ( 0.33431, 0.38239) MSU Line F1051 2 0.261759 0.001143 (0.237716, 0.285802) MSU Lin e F2001R 2 0.3261 0.0325 ( 0.3020, 0.3501) MSU Line F2002 2 0.31799 0.00807 ( 0.29395, 0.34203) MSU Line F2003 2 0.39026 0.00523 ( 0.36622, 0.41430) MSU Line F2004 2 0.3557 0.0184 ( 0.3316, 0.3797) MSU Line F2005 2 0.31841 0.00328 ( 0.29437, 0.34245) MSU Line F2006 2 0.23554 0.01211 ( 0.21150, 0.25958) MSU Line F2008 2 0.33907 0.00400 ( 0.31503, 0.36311) MSU Line F2009 2 0.32160 0.00726 ( 0.29756, 0.34565) MSU Line F 2011 2 0.3659 0.0258 ( 0.3418, 0.3899) MSU Line F2012 2 0.2853 0.0407 ( 0.2612, 0.3093) MSU Line F2014R 2 0.28355 0.01164 ( 0.25951, 0.30760) MSU Line F2015 2 0.37037 0.01172 ( 0.34632, 0.39441) MSU Line F2016 2 0.2896 0.0258 ( 0.2656, 0.3137) MSU Line F2018 2 0.36565 0.00972 ( 0.34161, 0.38969) MSU Line F2019 2 0.2857 0.0147 ( 0.2616, 0.3097) MSU Line F2020 2 0.2933 0.0223 ( 0.2693, 0.3174) MSU Line F2021 2 0.3105 0. 0398 ( 0.2865, 0.3346) MSU Line F2022 2 0.39717 0.01052 ( 0.37313, 0.42121) MSU Line F2024R 2 0.265141 0.001062 (0.241098, 0.289184) MSU Line F2025R 2 0.290139 0.000458 (0.266096, 0.314182) MSU Line F2027R 2 0.33946 0.00334 ( 0.31 542, 0.36350) MSU Line F2028R 2 0.32588 0.00991 ( 0.30184, 0.34992) MSU Line F2029R 2 0.3434 0.0207 ( 0.3194, 0.3675) MSU Line F2030 2 0.3032 0.0216 ( 0.2792, 0.3273) MSU Line F2031 2 0.31337 0.00342 ( 0.28933, 0.3374 1) MSU Line F2032 2 0.3737 0.0224 ( 0.3496, 0.3977) MSU Line F2033 2 0.27257 0.00316 ( 0.24853, 0.29661) MSU Line F2034 2 0.293135 0.001014 (0.269092, 0.317178) MSU Line F2035 2 0.3602 0.0429 ( 0.3361, 0.3842) MSU Line F2036 2 0.25297 0.01131 ( 0.22892, 0.27701) MSU Line F2037 2 0.31466 0.00151 ( 0.29062, 0.33871) MSU Line F2038 2 0.29723 0.00180 ( 0.27319, 0.32127) MSU Line F2039 2 0.412480 0.001059 (0.388437, 0.436524) MSU Line F2040 2 0.25763 0.01169 ( 0.23359, 0.28168) MSU Line F2041 2 0.34169 0.01260 ( 0.31764, 0.36573) MSU Line F2042 2 0.3711 0.0148 ( 0.3471, 0.3952) Red Ruby 2 0.3298 0.0148 ( 0.3058, 0.3538) VA09W - 188WS 2 0.27280 0. 01176 ( 0.24876, 0.29685) VA09W - 192WS 2 0.2654 0.0186 ( 0.2413, 0.2894) Pooled StDev = 0.0169801 Fisher Pairwise Comparisons Grouping Information Using the Fisher LSD Method and 95% Confidence C1 N Mean Grouping MSU Line F2039 2 0.412480 A MSU Line F2022 2 0.39717 A B MSU Line F2003 2 0.39026 A B C MSU Line F2032 2 0.3737 B C D MSU Line F2042 2 0.3711 B C D E MSU Line F2015 2 0.37037 B C D E MSU Line F2011 2 0.3659 B C D E F MSU Line F2018 2 0.36565 B C D E F MSU Line F1014 2 0.36218 C D E F G MSU Line F2035 2 0.3602 C D E F G MSU Line F1050 2 0.35835 C D E F G H Hopewell 2 0.3579 C D E F G H Ambassa dor 2 0.35762 C D E F G H MSU Line F2004 2 0.3557 D E F G H MSU Line F1027 2 0.35091 D E F G H I MSU Line F2029R 2 0.3434 D E F G H I J MSU Line F2041 2 0.34169 D E F G H I J K MSU Line F2027R 2 0.33946 E F G H I J K MSU Line F2008 2 0.33907 E F G H I J K Jupiter 2 0.3379 E F G H I J K MSU Line F1048 2 0.3376 E F G H I J K Cayuga 2 0.33490 F G H I J K L Red Ruby 2 0.3298 G H I J K L M MSU Line F2001R 2 0.3261 H I J K L M N MSU Line F2028R 2 0.32588 H I J K L M N MSU Line F1029 2 0.32584 H I J K L M N Caledonia 2 0.32529 H I J K L M N MSU Line F2009 2 0.32160 I J K L M N O MSU Line F2005 2 0.31841 I J K L M N O P MSU Line F2002 2 0.31799 I J K L M N O P MSU Line F2037 2 0.31466 J K L M N O P Q MSU Line F2031 2 0.31337 J K L M N O P Q Aubrey 2 0.31268 J K L M N O P Q MSU Line F2021 2 0.3105 J K L M N O P Q MSU Line F1012 2 0.3103 J K L M N O P Q MSU Line F1032R 2 0.30975 J K L M N O P Q MSU Line F1049 2 0.30909 K L M N O P Q MSU Line F2030 2 0.3032 L M N O P Q R MSU Line F1026R 2 0.30223 L M N O P Q R MSU Line F2038 2 0.29723 M N O P Q R S MSU Line F2020 2 0.2933 N O P Q R S T MSU Line F2034 2 0.293135 N O P Q R S T MSU Line F2025R 2 0.290139 O P Q R S T U MSU Line F2016 2 0.2896 O P Q R S T U MSU Line F1047 2 0.2882 O P Q R S T U MSU Line F2019 2 0.2857 P Q R S T U V MSU Line F2012 2 0.2853 P Q R S T U V MSU Line F2014R 2 0.28355 Q R S T U V W VA09W - 188WS 2 0.27280 R S T U V W MSU Line F2033 2 0.27257 R S T U V W VA09W - 192WS 2 0.2654 S T U V W X MSU Line F2024R 2 0.265141 S T U V W X MSU Line F1051 2 0.261759 T U V W X MSU Line F2040 2 0.25763 U V W X MSU Line F2036 2 0.25297 V W X MSU Line F1003R 2 0.24959 W X MSU Line F2006 2 0.23554 X Means that do not share a letter are significantly different. Table 13 .2. Raw Data for 2013 Bound Ferulic Acid Content One - way ANOVA: C2 versus C1 * ERROR * Cannot draw the interval plot. Interval plots are illegible with more than 45 intervals. Method Null hypothesis All means are equal Alternative hypothesis At least one mean is different Equal variances were assumed for the analysis. Analysis of Variance Source DF Adj SS Adj MS F - Value P - Value C1 56 176833 3157.7 10.95 0.000 Error 57 16434 288.3 Total 113 193268 Model Summary S R - sq R - sq(adj) R - sq(pred) 16.9801 91.50% 83.14% 65.99% Means C1 N Mean StDev 95% CI Ambassador 2 357.62 13.08 ( 333.58, 381.67) Aubrey 2 312.68 12.27 ( 288.64, 336.72) Caledonia 2 325.29 9.06 ( 301.24, 349.33) Cayuga 2 334.90 12.74 ( 310.86, 358.94) Hopewell 2 357.9 24.7 ( 333.9, 381.9) Jupiter 2 337.9 33.7 ( 313.9, 362.0) MSU Line F1003R 2 249.59 9.31 ( 225.55, 273.63) MSU Line F1012 2 310.3 21.9 ( 286.2, 334 .3) MSU Line F1014 2 362.18 5.62 ( 338.13, 386.22) MSU Line F1026R 2 302.23 5.51 ( 278.18, 326.27) MSU Line F1027 2 350.91 1.56 ( 326.87, 374.95) MSU Line F1029 2 325.84 7.18 ( 301.79, 349.88) MSU Line F1032R 2 309.75 3.12 ( 285.71, 333.79) MSU Line F1047 2 288.2 16.2 ( 264.2, 312.3) MSU Line F1048 2 337.6 26.8 ( 313.6, 361.7) MSU Line F1049 2 309.09 8.59 ( 285.05, 333.14) MSU Line F1050 2 358.35 13.03 ( 334.31, 382.39) MSU Line F1051 2 261.759 1.143 (237.716, 285.802) MSU Line F2001R 2 326.1 32.5 ( 302.0, 350.1) MSU Line F2002 2 317.99 8.07 ( 293.95, 342.03) MSU Line F2003 2 390.26 5.23 ( 366.22, 414.30) MSU Line F2004 2 3 55.7 18.4 ( 331.6, 379.7) MSU Line F2005 2 318.41 3.28 ( 294.37, 342.45) MSU Line F2006 2 235.54 12.11 ( 211.50, 259.58) MSU Line F2008 2 339.07 4.00 ( 315.03, 363.11) MSU Line F2009 2 321.60 7.26 ( 297.56, 345.65) MSU Line F2011 2 365.9 25.8 ( 341.8, 389.9) MSU Line F2012 2 285.3 40.7 ( 261.2, 309.3) MSU Line F2014R 2 283.55 11.64 ( 259.51, 307.60) MSU Line F2015 2 370.37 11.72 ( 346.32, 394.41) MSU Line F2016 2 289.6 25.8 ( 265.6, 313.7) MSU Line F2018 2 365.65 9.72 ( 341.61, 389.69) MSU Line F2019 2 285.7 14.7 ( 261.6, 309.7) MSU Line F2020 2 293.3 22.3 ( 269.3, 317.4) MSU Line F2021 2 310.5 39.8 ( 286.5, 334.6) MSU Line F2022 2 397.17 10.52 ( 373.13, 421.21) MSU Line F2024R 2 265.141 1.062 (241.098, 289.184) MSU Line F2025R 2 290.139 0.458 (266.096, 314.182) MSU Line F2027R 2 339.46 3.34 ( 315.42, 363.50) MSU Line F2028R 2 325.88 9.91 ( 301.84, 349.92) M SU Line F2029R 2 343.4 20.7 ( 319.4, 367.5) MSU Line F2030 2 303.2 21.6 ( 279.2, 327.3) MSU Line F2031 2 313.37 3.42 ( 289.33, 337.41) MSU Line F2032 2 373.7 22.4 ( 349.6, 397.7) MSU Line F2033 2 272.57 3.16 ( 248.53, 296.61) MSU Line F2034 2 293.135 1.014 (269.092, 317.178) MSU Line F2035 2 360.2 42.9 ( 336.1, 384.2) MSU Line F2036 2 252.97 11.31 ( 228.92, 277.01) MSU Line F2037 2 314.66 1.51 ( 290.62, 338.71) MSU Line F2038 2 297.23 1.80 ( 273.19, 321.27) MSU Line F2039 2 412.480 1.059 (388.437, 436.524) MSU Line F2040 2 257.63 11.69 ( 233.59, 281.68) MSU Line F2041 2 341.69 12.60 ( 317.64, 365.73) MSU Line F2042 2 371.1 14.8 ( 347.1, 395.2) Red Ruby 2 329.8 14.8 ( 305.8, 353.8) VA09W - 188WS 2 272.80 11.76 ( 248.76, 296.85) VA09W - 192WS 2 265.4 18.6 ( 241.3, 289.4) Pooled StDev = 16.9801 Fisher Pairwise Comparisons Grouping Information Using the Fisher LSD Method and 95% Confidence C1 N Mean Grouping MSU Line F2039 2 412.480 A MSU Line F2022 2 397.17 A B MSU Line F2003 2 390.26 A B C MSU Line F2032 2 373.7 B C D MSU Line F2042 2 371.1 B C D E MSU L ine F2015 2 370.37 B C D E MSU Line F2011 2 365.9 B C D E F MSU Line F2018 2 365.65 B C D E F MSU Line F1014 2 362.18 C D E F G MSU Line F2035 2 360.2 C D E F G MSU Line F1050 2 358.35 C D E F G H Hopewell 2 357.9 C D E F G H Ambassador 2 357.62 C D E F G H MSU Line F2004 2 355.7 D E F G H MSU Line F1027 2 350.91 D E F G H I MSU Line F2029R 2 343.4 D E F G H I J MSU Line F204 1 2 341.69 D E F G H I J K MSU Line F2027R 2 339.46 E F G H I J K MSU Line F2008 2 339.07 E F G H I J K Jupiter 2 337.9 E F G H I J K MSU Line F1048 2 337.6 E F G H I J K Cayuga 2 334.90 F G H I J K L Red Ruby 2 329.8 G H I J K L M MSU Line F2001R 2 326.1 H I J K L M N MSU Line F2028R 2 325.88 H I J K L M N MSU Line F1029 2 325.84 H I J K L M N Caledonia 2 325.29 H I J K L M N MSU Line F2009 2 321.60 I J K L M N O MSU Line F2005 2 318.41 I J K L M N O P MSU Line F2002 2 317.99 I J K L M N O P MSU Line F2037 2 314.66 J K L M N O P Q MSU Line F2031 2 313.37 J K L M N O P Q Aubrey 2 312.68 J K L M N O P Q MSU Line F2021 2 310.5 J K L M N O P Q MSU Line F101 2 2 310.3 J K L M N O P Q MSU Line F1032R 2 309.75 J K L M N O P Q MSU Line F1049 2 309.09 K L M N O P Q MSU Line F2030 2 303.2 L M N O P Q R MSU Line F1026R 2 302.23 L M N O P Q R MSU Line F2038 2 297.23 M N O P Q R S MSU Line F2020 2 293.3 N O P Q R S T MSU Line F2034 2 293.135 N O P Q R S T M SU Line F2025R 2 290.139 O P Q R S T U MSU Line F2016 2 289.6 O P Q R S T U MSU Line F1047 2 288.2 O P Q R S T U MSU Line F2019 2 285.7 P Q R S T U V MSU Line F2012 2 285.3 P Q R S T U V MSU Line F2014R 2 283.55 Q R S T U V W VA09W - 188WS 2 272.80 R S T U V W MSU Line F2033 2 272.57 R S T U V W VA09W - 192WS 2 265.4 S T U V W X MSU Line F2024R 2 265.141 S T U V W X MSU Line F1051 2 261.759 T U V W X MSU Line F2040 2 257.63 U V W X MSU Line F2036 2 252.97 V W X MSU Line F1003R 2 249.59 W X MSU Line F2006 2 235.54 X Means that do not share a letter are significantly different. Table 13 .3. Raw Data for 2013 Free Ferulic Acid Content One - way ANOVA: C2 versus C1 * ERROR * Cannot draw the interval plot. Interval plots are illegible with more than 45 intervals. Method Null hypothesis All means are equal Alternative hypothesis At least one mean is different Equal variances were assumed for the analysis. Analysis of Variance Source DF Adj SS Adj MS F - Value P - Value C1 56 3251.3 58.06 3.75 0.000 Error 57 881.4 15.46 Total 113 4132.8 Model Summary S R - sq R - sq(adj) R - sq(pr ed) 3.93240 78.67% 57.72% 14.69% Means C1 N Mean StDev 95% CI Ambassador 2 21.40 2.83 ( 15.83, 26.97) Aubrey 2 16.20 2.12 ( 10.63, 21.77) Caledonia 2 14.10 2.12 ( 8.53, 19.67) C ayuga 2 19.40 2.83 ( 13.83, 24.97) Hopewell 2 20.80 4.95 ( 15.23, 26.37) Jupiter 2 19.70 7.07 ( 14.13, 25.27) MSU Line F1003R 2 18.40 2.83 ( 12.83, 23.97) MSU Line F1012 2 17.00 4.95 ( 11.43, 22.57) MSU Line F1014 2 13.90 3.54 ( 8.33, 19.47) MSU Line F1026R 2 13.50 1.41 ( 7.93, 19.07) MSU Line F1027 2 13.800 0.283 ( 8.232, 19.368) MSU Line F1029 2 13.40 1.41 ( 7.83, 18.97) MSU Line F1032R 2 11.900 0.707 ( 6.33 2, 17.468) MSU Line F1047 2 17.40 4.24 ( 11.83, 22.97) MSU Line F1048 2 27.50 5.66 ( 21.93, 33.07) MSU Line F1049 2 12.50 2.12 ( 6.93, 18.07) MSU Line F1050 2 32.10 2.83 ( 26.53, 37.67) MSU Line F1051 2 21.700 0.283 (1 6.132, 27.268) MSU Line F2001R 2 23.50 7.07 ( 17.93, 29.07) MSU Line F2002 2 17.40 2.12 ( 11.83, 22.97) MSU Line F2003 2 16.300 0.707 (10.732, 21.868) MSU Line F2004 2 26.00 3.54 ( 20.43, 31.57) MSU Line F20 05 2 25.700 0.707 (20.132, 31.268) MSU Line F2006 2 21.90 3.54 ( 16.33, 27.47) MSU Line F2008 2 33.00 7.78 ( 27.43, 38.57) MSU Line F2009 2 18.50 1.41 ( 12.93, 24.07) MSU Line F2011 2 20.90 4.95 ( 15.33, 26.47) MSU Line F2012 2 19.90 9.90 ( 14.33, 25.47) MSU Line F2014R 2 18.40 2.83 ( 12.83, 23.97) MSU Line F2015 2 16.90 2.12 ( 11.33, 22.47) MSU Line F2016 2 19.10 6.36 ( 13.53, 24.67) MSU Line F2018 2 26.550 0.212 (20.982, 32.118) MSU Line F2019 2 14.00 3.54 ( 8.43, 19.57) MSU Line F2020 2 12.00 4.95 ( 6.43, 17.57) MSU Line F2021 2 26.80 9.19 ( 21.23, 32.37) MSU Line F2022 2 15.20 2.12 ( 9.63, 20.77) MSU Line F2024R 2 15.850 0.354 (10.282, 21.418) MSU Line F2025R 2 18.600 0.141 (13.032, 24.168) MSU Line F2027R 2 20.000 0.707 (14.432, 25.568) MSU Line F2028R 2 20.20 2.12 ( 14.63, 25.77) MSU Line F2029R 2 29.80 4.24 ( 24.23, 35.37) MSU Line F2030 2 25.00 4.95 ( 19.43, 30. 57) MSU Line F2031 2 14.100 0.707 ( 8.532, 19.668) MSU Line F2032 2 18.60 4.24 ( 13.03, 24.17) MSU Line F2033 2 26.200 0.707 (20.632, 31.768) MSU Line F2034 2 11.700 0.283 ( 6.132, 17.268) MSU Line F2035 2 18.50 8.49 ( 12.93, 24.07) MSU Line F2036 2 15.10 3.54 ( 9.53, 20.67) MSU Line F2037 2 19.900 0.424 (14.332, 25.468) MSU Line F2038 2 11.950 0.495 ( 6.382, 17.518) MSU Line F2039 2 12.900 0.283 ( 7.332, 18.468) MSU Line F2040 2 22.00 3.54 ( 16. 43, 27.57) MSU Line F2041 2 23.50 2.83 ( 17.93, 29.07) MSU Line F2042 2 27.60 2.83 ( 22.03, 33.17) Red Ruby 2 27.80 3.54 ( 22.23, 33.37) VA09W - 188WS 2 16.60 2.83 ( 11.03, 22.17) VA09W - 192WS 2 17.20 4.95 ( 11.63, 22.77) Pooled StDev = 3.93240 Fisher Pairwise Comparisons Grouping Information Using the Fisher LSD Method and 95% Confidence C1 N Mean Grouping MSU Line F2008 2 33.00 A MSU Line F1050 2 32.10 A B MSU Line F2029R 2 29.80 A B C Red Ruby 2 27.80 A B C D MSU Line F2042 2 27.60 A B C D E MSU Line F1048 2 27.50 A B C D E F MSU Line F2021 2 26.80 A B C D E F G MSU Line F2018 2 26.550 A B C D E F G MSU Line F2033 2 26.200 A B C D E F G H MSU Line F2004 2 26.00 A B C D E F G H MSU Line F2005 2 25.700 A B C D E F G H MSU Line F2030 2 25.00 B C D E F G H I MSU Line F2041 2 23.50 C D E F G H I J MSU Line F2001R 2 23.50 C D E F G H I J MSU Line F2040 2 22.00 C D E F G H I J K MSU Line F2006 2 21.90 D E F G H I J K L MSU Line F1051 2 21.700 D E F G H I J K L M Ambassador 2 21.40 D E F G H I J K L M N MSU Line F2011 2 20.90 D E F G H I J K L M N O Hopewell 2 20.80 D E F G H I J K L M N O MSU Line F2028R 2 20.20 D E F G H I J K L M N O P MSU Line F2027R 2 20.000 D E F G H I J K L M N O P MSU Line F2037 2 19.900 E F G H I J K L M N O P MSU Line F2012 2 19.90 E F G H I J K L M N O P Jupiter 2 19.70 F G H I J K L M N O P Q Cayuga 2 19.40 G H I J K L M N O P Q R MSU Line F2016 2 19.10 G H I J K L M N O P Q R MSU Line F 2032 2 18.60 H I J K L M N O P Q R MSU Line F2025R 2 18.600 H I J K L M N O P Q R MSU Line F2035 2 18.50 H I J K L M N O P Q R MSU Line F2009 2 18.50 H I J K L M N O P Q R MSU Line F 2014R 2 18.40 H I J K L M N O P Q R MSU Line F1003R 2 18.40 H I J K L M N O P Q R MSU Line F2002 2 17.40 I J K L M N O P Q R MSU Line F1047 2 17.40 I J K L M N O P Q R VA09W - 192W S 2 17.20 I J K L M N O P Q R MSU Line F1012 2 17.00 J K L M N O P Q R MSU Line F2015 2 16.90 J K L M N O P Q R VA09W - 188WS 2 16.60 J K L M N O P Q R MSU Line F 2003 2 16.300 J K L M N O P Q R Aubrey 2 16.20 J K L M N O P Q R MSU Line F2024R 2 15.850 J K L M N O P Q R MSU Line F2022 2 15.20 K L M N O P Q R MSU Line F 2036 2 15.10 K L M N O P Q R MSU Line F2031 2 14.100 L M N O P Q R Caledonia 2 14.10 L M N O P Q R MSU Line F2019 2 14.00 M N O P Q R MSU Line F1014 2 13.90 M N O P Q R MSU Line F1027 2 13.800 N O P Q R MSU Line F1026R 2 13.50 O P Q R MSU Line F1029 2 13.40 O P Q R MSU Line F2039 2 12.900 P Q R MSU Line F1049 2 12.50 P Q R MSU Line F2020 2 12.00 Q R MSU Line F2038 2 11.950 Q R MSU Line F1032R 2 11.900 Q R MSU Line F2034 2 11.700 R Means that do not share a letter are significantly different. Figure 13 .4. Raw Data for 2014 Total Ferulic Acid Content One - way ANOVA: C2 versus C1 Method Null hypothesis All means are equal Alternative hypothesis At least one mean is different Equal variances were assumed for the analysis. Analysis of Variance Source DF Adj SS Adj MS F - Value P - Value C1 38 113361 2983 1.92 0.023 Error 39 60519 1552 Total 77 173880 Model Summary S R - sq R - sq(adj) R - sq(pred) 39.3924 65.20% 31.28% 0.00% Means C1 N Mean StDev 95% CI Ambassador 2 304.72 2.87 ( 248.38, 361.07) Aubrey 2 302.9 23.0 ( 246.6, 359.2) F0013R 2 330.3 33.2 ( 274.0, 386.7) F2010 2 351.0 20.7 ( 294.7, 407.4) Jupiter 2 297.7 34.0 ( 241.3, 354.0) MSU Line F1026R 2 370.29 7.27 ( 313.95, 426.63) MSU Line F1027 2 352.693 1.145 (296.351, 409.034) MSU Line F1029 2 399.3 49.4 ( 342.9, 455.6) MSU Line F1047 2 350.7 60.8 ( 294.3, 407 .0) MSU Line F1048 2 388.02 9.70 ( 331.68, 444.36) MSU Line F1049 2 355.637 1.296 (299.296, 411.979) MSU Line F2002 2 342.8 65.0 ( 286.5, 399.2) MSU Line F2003 2 414.5 45.5 ( 358.2, 470.9) MSU Line F2005 2 303.672 0.496 (247.330, 360.013) MSU Line F2008 2 372.0 16.4 ( 315.7, 428.4) MSU Line F2009 2 386.6 19.0 ( 330.3, 443.0) MSU Line F2012 2 414.857 0.582 (358.516, 471.198) MSU Line F2014R 2 434.6 114.2 ( 378.2, 490.9) MSU Line F 2015 2 340.90 6.15 ( 284.55, 397.24) MSU Line F2016 2 375.44 9.23 ( 319.10, 431.78) MSU Line F2018 2 344.4 39.4 ( 288.1, 400.7) MSU Line F2019 2 329.2 31.2 ( 272.8, 385.5) MSU Line F2020 2 356.30 3.11 ( 299.96, 412.64) MSU Line F2021 2 348.5 26.9 ( 292.2, 404.9) MSU Line F2022 2 340.8 53.3 ( 284.5, 397.2) MSU Line F2024R 2 363.3 26.0 ( 306.9, 419.6) MSU Line F2028R 2 442.2 100.9 ( 385.9, 498.6) MSU Li ne F2029R 2 429.2 28.7 ( 372.9, 485.6) MSU Line F2030 2 389.8 82.9 ( 333.5, 446.2) MSU Line F2031 2 362.9 23.0 ( 306.6, 419.3) MSU Line F2033 2 306.77 7.21 ( 250.43, 363.11) MSU Line F2034 2 372.9 28.6 ( 316 .6, 429.3) MSU Line F2037 2 353.15 2.52 ( 296.81, 409.49) MSU Line F2038 2 399.1 14.3 ( 342.7, 455.4) MSU Line F2039 2 428.5 22.5 ( 372.2, 484.9) MSU Line F2042 2 325.24 10.72 ( 268.90, 381.58) Unnamed 1 2 374.4 32.5 ( 318.1, 430.7) Unnamed 2 2 347.03 6.06 ( 290.69, 403.38) Unnamed 3 2 406.5 54.1 ( 350.2, 462.8) Pooled StDev = 39.3924 Fisher Pairwise Comparisons Grouping Information Using the Fisher LSD Method and 95% Confidence C1 N Mean Grouping MSU Line F2028R 2 442.2 A MSU Line F2014R 2 434.6 A B MSU Line F2029R 2 429.2 A B C MSU Line F2039 2 428.5 A B C MSU Line F2012 2 414.857 A B C D MSU Line F2003 2 414.5 A B C D Unnamed 3 2 406.5 A B C D E MSU Line F1029 2 399.3 A B C D E F MSU Line F2038 2 399.1 A B C D E F MSU Line F2030 2 389.8 A B C D E F MSU Line F1048 2 388.02 A B C D E F MSU Line F2009 2 386.6 A B C D E F MSU Line F2016 2 375.44 A B C D E F G Unnamed 1 2 374.4 A B C D E F G MSU Line F2034 2 372.9 A B C D E F G MSU Line F2008 2 372.0 A B C D E F G MSU Line F1026R 2 370.29 A B C D E F G MSU Line F2024R 2 363.3 A B C D E F G MSU Line F2031 2 362.9 A B C D E F G MSU Line F2020 2 356.30 B C D E F G MSU Line F1049 2 355.637 B C D E F G MSU Line F2037 2 353.15 C D E F G MSU Line F1027 2 352.693 C D E F G F2010 2 351.0 C D E F G MS U Line F1047 2 350.7 C D E F G MSU Line F2021 2 348.5 D E F G Unnamed 2 2 347.03 D E F G MSU Line F2018 2 344.4 D E F G MSU Line F2002 2 342.8 D E F G MSU Line F2015 2 3 40.90 D E F G MSU Line F2022 2 340.8 D E F G F0013R 2 330.3 E F G MSU Line F2019 2 329.2 E F G MSU Line F2042 2 325.24 F G MSU Line F2033 2 306.77 G Ambassador 2 304.72 G MSU Line F2005 2 303.672 G Aubrey 2 302.9 G Jupiter 2 297.7 G Means that do not share a letter are significantly different. Table 13 .5. Raw Data for 2014 Bound Ferulic Acid Content One - way ANOVA: C2 versus C1 Method Null hypothesis All means are equal Alternative hypothesis At least one mean is different Equal variances were assumed for the analysis. Analysis of Va riance Source DF Adj SS Adj MS F - Value P - Value C1 38 110382 2905 2.05 0.014 Error 39 55308 1418 Total 77 165690 Model Summary S R - sq R - sq(adj) R - sq(pred) 37.6584 66.62% 34.10% 0.00% Means C1 N Mean StDev 95% CI Ambassador 2 323.536 0.863 (269.675, 377.398) Aubrey 2 333.81 6.01 ( 279.95, 387.67) F0013R 2 368.2 37.8 ( 314.3, 422.0) F2010 2 371.01 8.69 ( 317. 15, 424.87) Jupiter 2 316.3 36.3 ( 262.4, 370.1) MSU Line F1026R 2 388.48 3.85 ( 334.62, 442.34) MSU Line F1027 2 375.06 12.52 ( 321.20, 428.93) MSU Line F1029 2 418.5 48.5 ( 364.7, 472.4) MSU Line F1047 2 3 86.6 68.7 ( 332.7, 440.5) MSU Line F1048 2 403.12 9.52 ( 349.26, 456.98) MSU Line F1049 2 373.20 2.09 ( 319.34, 427.06) MSU Line F2002 2 367.9 49.6 ( 314.1, 421.8) MSU Line F2003 2 432.0 45.2 ( 378.1, 485.8) MSU Line F2005 2 317.748 0.742 (263.886, 371.609) MSU Line F2008 2 390.3 14.9 ( 336.4, 444.2) MSU Line F2009 2 402.7 19.5 ( 348.9, 456.6) MSU Line F2012 2 429.427 0.105 (375.566, 483.288) MSU Line F2014R 2 457.5 113.7 ( 4 03.6, 511.4) MSU Line F2015 2 356.32 7.31 ( 302.46, 410.18) MSU Line F2016 2 396.15 9.65 ( 342.29, 450.01) MSU Line F2018 2 367.6 35.9 ( 313.8, 421.5) MSU Line F2019 2 353.4 30.1 ( 299.6, 407.3) MSU Line F2020 2 372.68 1.74 ( 318.82, 426.55) MSU Line F2021 2 381.4 20.8 ( 327.6, 435.3) MSU Line F2022 2 360.3 51.1 ( 306.4, 414.1) MSU Line F2024R 2 381.5 27.3 ( 327.7, 435.4) MSU Line F2028R 2 467.2 93.6 ( 413.4, 521.1) MSU Line F2029R 2 451.7 26.2 ( 397.8, 505.6) MSU Line F2030 2 421.1 68.0 ( 367.2, 474.9) MSU Line F2031 2 384.9 19.9 ( 331.0, 438.7) MSU Line F2033 2 338.21 11.21 ( 284.35, 392.07) MSU Line F2034 2 396.9 20.1 ( 343.1, 450.8) MSU Line F2037 2 370.44 2.64 ( 316.58, 424.30) MSU Line F2038 2 413.35 9.07 ( 359.49, 467.21) MSU Line F2039 2 456.5 41.2 ( 402.7, 510.4) MSU Line F2042 2 346.3 14.7 ( 292.4, 400.2 ) Unnamed 1 2 391.3 35.7 ( 337.4, 445.1) Unnamed 2 2 365.751 0.443 (311.890, 419.612) Unnamed 3 2 422.4 51.8 ( 368.6, 476.3) Pooled StDev = 37.6584 Fisher Pairwise Comparisons Grouping Information Using the Fisher LSD Method and 95% Confidence C1 N Mean Grouping MSU Line F2028R 2 467.2 A MSU Line F2014R 2 457.5 A B MSU Line F2039 2 456.5 A B MSU Line F2029R 2 451.7 A B MSU Line F2003 2 432.0 A B C MSU Line F2012 2 429.427 A B C D Unnamed 3 2 422.4 A B C D E MSU Line F2030 2 421.1 A B C D E MSU Line F1029 2 418.5 A B C D E MSU Line F2038 2 413.35 A B C D E F MSU Line F1048 2 403.12 A B C D E F G MSU Line F2009 2 402.7 A B C D E F G MSU Line F2034 2 396.9 A B C D E F G H MSU Line F2016 2 396.15 A B C D E F G H Unnamed 1 2 391.3 A B C D E F G H I MSU Line F2008 2 390.3 B C D E F G H I MSU Line F1026R 2 388.48 B C D E F G H I MSU Line F1047 2 386.6 B C D E F G H I MSU Line F2031 2 384.9 B C D E F G H I MSU Line F2024R 2 381.5 B C D E F G H I MSU Line F2021 2 381.4 B C D E F G H I MSU Line F1027 2 375.06 C D E F G H I MSU Line F1049 2 373.20 C D E F G H I MSU Line F2020 2 372.68 C D E F G H I F2010 2 371.01 C D E F G H I MSU Line F2037 2 370.44 C D E F G H I F0013R 2 368.2 C D E F G H I MSU Line F2002 2 367.9 C D E F G H I Table 13.5 MSU Line F2018 2 367.6 C D E F G H I Unnamed 2 2 365.751 C D E F G H I MSU Line F2022 2 360.3 C D E F G H I MSU Line F2015 2 356.32 C D E F G H I MSU Line F2019 2 353.4 D E F G H I MSU Line F 2042 2 346.3 E F G H I MSU Line F2033 2 338.21 F G H I Aubrey 2 333.81 G H I Ambassador 2 323.536 H I MSU Line F2005 2 317.748 I Jupiter 2 316.3 I Means that do not share a letter are significantly different. Table 13 .6. Raw Data for 2014 Free Ferulic Acid Content One - way ANOVA: C2 versus C1 Method Null hypothesis All means are equal Alternative hypothesis At least one mean is different Equal variances were assumed for the analysis. Analysis of Variance Source DF Adj SS Adj MS F - Value P - Value C1 38 2867 75.46 1.62 0.069 Error 39 1816 46.57 Total 77 46 84 Model Summary S R - sq R - sq(adj) R - sq(pred) 6.82416 61.22% 23.44% 0.00% Means C1 N Mean StDev 95% CI Ambassador 2 18.81 2.01 ( 9.05, 28.57) Aubrey 2 30.9 17.0 ( 21.1 , 40.7) F0013R 2 37.82 4.65 ( 28.06, 47.58) F2010 2 19.98 12.04 ( 10.22, 29.74) Jupiter 2 18.60 2.31 ( 8.84, 28.36) MSU Line F1026R 2 18.19 3.42 ( 8.43, 27.95) MSU Line F1027 2 22.37 11.37 ( 12.61, 32.13) MSU Line F1029 2 19.255 0.844 ( 9.494, 29.015) MSU Line F1047 2 35.95 7.83 ( 26.19, 45.71) MSU Line F1048 2 15.096 0.181 ( 5.336, 24.856) MSU Line F1049 2 17.563 0.796 ( 7.803, 27.324) MSU Line F2002 2 25.1 15.4 ( 15.3, 34.9) MSU Line F2003 2 17.473 0.288 ( 7.713, 27.233) MSU Line F2005 2 14.076 1.238 ( 4.316, 23.836) MSU Line F2008 2 18.27 1.58 ( 8.51, 28.04) MSU Line F2009 2 16.083 0.516 ( 6.322, 25.843) MSU Line F2012 2 14.570 0.687 ( 4.810, 24.330) MSU Line F2014R 2 22.930 0.526 (13.169, 32.690) MSU Line F2015 2 15.426 1.162 ( 5.666, 25.186) MSU Line F2016 2 20.709 0.419 (10.948, 30.469) MSU Line F2018 2 23.23 3.51 ( 13.47, 32.99) MSU Line F2019 2 24.231 1.099 (14.471, 33.991) MSU Line F2020 2 16.38 4.84 ( 6.62, 26.14) MSU Line F2021 2 32.90 6.19 ( 23.14, 42.66) MSU Line F2022 2 19.44 2.21 ( 9.68, 29.20) M SU Line F2024R 2 18.244 1.289 ( 8.484, 28.005) MSU Line F2028R 2 24.99 7.34 ( 15.23, 34.75) MSU Line F2029R 2 22.47 2.54 ( 12.71, 32.23) MSU Line F2030 2 31.2 15.0 ( 21.5, 41.0) MSU Line F2031 2 21.93 3.11 ( 12.17, 31.69) MSU Line F2033 2 31.44 4.00 ( 21.68, 41.20) MSU Line F2034 2 24.00 8.55 ( 14.24, 33.76) MSU Line F2037 2 17.2940 0.1247 (7.5336, 27.0543) MSU Line F2038 2 14.27 5.20 ( 4.51, 24.0 3) MSU Line F2039 2 28.0 18.7 ( 18.2, 37.8) MSU Line F2042 2 21.06 3.96 ( 11.29, 30.82) Unnamed 1 2 16.89 3.22 ( 7.13, 26.65) Unnamed 2 2 18.72 5.62 ( 8.96, 28.48) Unnamed 3 2 15.93 2. 21 ( 6.17, 25.69) Pooled StDev = 6.82416 Fisher Pairwise Comparisons Grouping Information Using the Fisher LSD Method and 95% Confidence C1 N Mean Grouping F0013R 2 37.82 A MSU Line F1047 2 35.95 A B MSU Li ne F2021 2 32.90 A B C MSU Line F2033 2 31.44 A B C D MSU Line F2030 2 31.2 A B C D E Aubrey 2 30.9 A B C D E F MSU Line F2039 2 28.0 A B C D E F G MSU Line F2002 2 25.1 A B C D E F G H MSU Line F2028R 2 24.99 A B C D E F G H MSU Line F2019 2 24.231 A B C D E F G H MSU Line F2034 2 24.00 B C D E F G H MSU Line F2018 2 23.23 B C D E F G H MSU Line F2014R 2 22.930 B C D E F G H MSU Line F2029R 2 22.47 B C D E F G H MSU Line F1027 2 22.37 B C D E F G H MSU Line F2031 2 21.93 C D E F G H MSU Line F2042 2 21.06 C D E F G H MSU Line F2016 2 20.709 C D E F G H F2010 2 19.98 C D E F G H MSU Line F2022 2 19.44 C D E F G H MSU Line F1029 2 19.255 C D E F G H Ambassador 2 18.81 D E F G H Unnamed 2 2 18.72 D E F G H Jupiter 2 18.60 D E F G H MSU Line F2008 2 18.27 D E F G H MSU Line F2024R 2 18.2 44 D E F G H MSU Line F1026R 2 18.19 D E F G H MSU Line F1049 2 17.563 E F G H MSU Line F2003 2 17.473 E F G H MSU Line F2037 2 17.2940 F G H Unnamed 1 2 16.89 G H MSU Line F2020 2 16.38 G H MSU Line F2009 2 16.083 G H Unnamed 3 2 15.93 G H MSU Line F2015 2 15.426 G H MSU Line F1048 2 15.096 G H MSU Line F2012 2 14.570 G H MSU Line F2038 2 14.27 G H MSU Line F2005 2 14.076 H Means that do not share a letter are significantly different. Table 13 .7. Raw Data for 2013 Total Phenolic Acid Content One - way ANOVA: C2 v ersus C1 * ERROR * Cannot draw the interval plot. Interval plots are illegible with more than 45 intervals. * NOTE * Cannot draw the interval plot for the Fisher procedure. Interval plots for comparisons are illegible with more than 45 intervals. Method Null hypothesis All means are equal Alternative hypothesis At least one mean is different Equal variances were assumed for the analysis. Analysis of Variance Source DF Adj SS Adj MS F - Value P - Value C1 56 90.240 1.61142 42.34 0.000 Error 114 4.338 0.03806 Total 170 94.578 Model Summary S R - sq R - sq(adj) R - sq(pred) 0.195078 95.41% 93.16% 89.68% Means C1 N Mean StDev 95 % CI E0028 3 4.700 0.265 ( 4.477, 4.923) E1007R 3 4.400 0.173 ( 4.177, 4.623) E5011 3 7.0667 0.1155 (6.8436, 7.2898) F1003R 3 4.000 0.000 ( 3.777, 4.223) F1012 3 4.800 0.436 ( 4.577, 5.023) F1014 3 6.800 0.173 ( 6. 577, 7.023) F1026R 3 4.567 0.208 ( 4.344, 4.790) F1027 3 4.1000 0.1000 (3.8769, 4.3231) F1029 3 5.567 0.231 ( 5.344, 5.790) F1032R 3 4.900 0.000 ( 4.677, 5.123) F1047 3 3.8500 0.0500 (3.6269, 4.0731) F1048 3 4.750 0.2 50 ( 4.527, 4.973) F1049 3 4.000 0.265 ( 3.777, 4.223) F1050 3 4.5500 0.0500 (4.3269, 4.7731) F1051 3 3.5500 0.0500 (3.3269, 3.7731) F2001R 3 5.1500 0.0500 (4.9269, 5.3731) F2002 3 4.4500 0.0500 (4.2269, 4.6731) F2003 3 5.967 0.208 ( 5.744, 6.190) F2004 3 4.667 0.231 ( 4.444, 4.890) F2005 3 4.2500 0.0500 (4.0269, 4.4731) F2006 3 5.2000 0.1000 (4.9769, 5.4231) F2008 3 4.800 0.265 ( 4.577, 5.023) F2009 3 4.600 0.361 ( 4.377, 4.823) F2011 3 5.300 0.361 ( 5.077, 5.523) F2012 3 4.000 0.173 ( 3.777, 4.223) F2014R 3 5.000 0.173 ( 4.777, 5.223) F2015 3 4.5500 0.1500 (4.3269, 4.7731) F2016 3 4.3500 0.0500 (4.1269, 4.5731) F2018 3 4.200 0.300 ( 3.977, 4.423) F2019 3 3.9500 0.1500 (3.7269, 4.1731) F2020 3 3.8500 0.1500 (3.6269, 4.0731) F2021 3 5.200 0.529 ( 4.977, 5.423) F2022 3 5.100 0.000 ( 4.877, 5.323) F2024R 3 4.4500 0.0500 (4.2269, 4.6731) F2025R 3 6.1000 0.1000 (5.8769, 6.3231) F2027R 3 5.167 0.306 ( 4.944, 5.390) F2028R 3 4.3500 0.1500 (4.1269, 4.5731) F2029R 3 5.600 0.173 ( 5.377, 5.823) F2030 3 6.3000 0.1000 (6.0769, 6.5231) F2031 3 4.867 0.321 ( 4.644, 5.090) F2032 3 3.8500 0.1500 (3.6269, 4.0731) F2033 3 4.700 0.265 ( 4.477, 4.923) F2034 3 4.3500 0.0500 (4.1269, 4.5731) F2035 3 5.1500 0.0500 (4.9269, 5.3731) F2036 3 4.2500 0.1500 (4.0269, 4.4731) F2037 3 3.667 0.321 ( 3.444, 3.890) F2038 3 5.000 0.200 ( 4.777, 5.223) F2039 3 5.3500 0.1500 (5.1269, 5.5731) F2040 3 5.0000 0.1000 (4.7769, 5.2231) F2041 3 4.3500 0.0500 (4.1269, 4.5731) F2042 3 4.0500 0.1500 (3.8269, 4.2731) I5440 3 4.8500 0.1500 ( 4.6269, 5.0731) I7067 3 3.9500 0.0500 (3.7269, 4.1731) I7127 3 5.1500 0.1500 (4.9269, 5.3731) I7826 3 4.9500 0.0500 (4.7269, 5.1731) I9339 3 4.1500 0.0500 (3.9269, 4.3731) I9340 3 4.400 0.000 ( 4.177, 4.623) Pooled StDev = 0.19 5078 Fisher Pairwise Comparisons Grouping Information Using the Fisher LSD Method and 95% Confidence C1 N Mean Grouping E5011 3 7.0667 A F1014 3 6.800 A F2030 3 6.3000 B F2025R 3 6.1000 B C F2003 3 5.967 C F2029R 3 5.600 D F1029 3 5.567 D F2039 3 5.3500 D E F2011 3 5.300 D E F F2021 3 5.200 E F G F2006 3 5.2000 E F G F2027R 3 5.167 E F G H I7127 3 5.1500 E F G H I F2035 3 5.1500 E F G H I F2001R 3 5.1500 E F G H I F2022 3 5.100 E F G H I J F2040 3 5.0000 F G H I J K F2038 3 5.000 F G H I J K F2014R 3 5.000 F G H I J K I7826 3 4.9500 G H I J K L F1032R 3 4.900 G H I J K L M F2031 3 4.867 H I J K L M N I5440 3 4.8500 I J K L M N O F2008 3 4.800 J K L M N O F1012 3 4.800 J K L M N O F1048 3 4.750 K L M N O P F2033 3 4.700 K L M N O P Q E0028 3 4.700 K L M N O P Q F2004 3 4.667 L M N O P Q F2009 3 4.600 M N O P Q R F1026R 3 4.567 N O P Q R F2015 3 4.5500 O P Q R S F1050 3 4.5500 O P Q R S F2024R 3 4.4500 P Q R S T F2 002 3 4.4500 P Q R S T I9340 3 4.400 Q R S T U E1007R 3 4.400 Q R S T U F2034 3 4.3500 R S T U V F2041 3 4.3 500 R S T U V F2016 3 4.3500 R S T U V F2028R 3 4.3500 R S T U V F2036 3 4.2500 S T U V W F2005 3 4.250 0 S T U V W F2018 3 4.200 T U V W I9339 3 4.1500 T U V W X F1027 3 4.1000 U V W X F2042 3 4.0500 V W X F2012 3 4.000 W X F1049 3 4.000 W X F1003R 3 4.000 W X I7067 3 3.9500 W X Y F2019 3 3.9500 W X Y F2032 3 3.8500 X Y Z F2020 3 3.8500 X Y Z F1047 3 3.8500 X Y Z F2037 3 3.667 Y Z F1051 3 3.5500 Z Means that do not share a letter are significantly different. Table 13 .8. Raw Data for 2013 Bound Phenolic Content One - way ANOVA: C2 versus C1 * ERROR * Cannot draw the interval plot. Interval plots are illegible with more than 45 intervals. * NOTE * Cannot draw the interval plot for the Fisher procedure. Interval plots for comparisons are illegible with more than 45 intervals. Method Null hypothesis All means are equal Alternative hypothesis At least one mean is different Significance le Equal variances were assumed for the analysis. Analysis of Variance Source DF Adj SS Adj MS F - Value P - Value C1 56 6.517 0.11637 2.54 0.000 Error 114 5.213 0.04573 Total 170 11.730 Model Summary S R - sq R - sq(adj) R - sq(pred) 0.213838 55.56% 33.73% 0.01% Means C1 N Mean StDev 95% CI E0028 3 2.1138 0.0873 (1.8692, 2.3584) E1007R 3 1.9314 0.1413 (1.6868, 2.1759) E5011 3 1.846 0.174 ( 1.601, 2.0 90) F1003R 3 2.1276 0.1727 (1.8830, 2.3722) F1012 3 2.340 0.346 ( 2.095, 2.584) F1014 3 2.041 0.270 ( 1.796, 2.285) F1026R 3 2.121 0.190 ( 1.877, 2.366) F1027 3 2.2723 0.0714 (2.0277, 2.5168) F1029 3 2.1279 0.0282 (1.88 34, 2.3725) F1032R 3 2.0670 0.0262 (1.8225, 2.3116) F1047 3 2.093 0.360 ( 1.849, 2.338) F1048 3 2.328 0.436 ( 2.083, 2.572) F1049 3 2.450 0.324 ( 2.205, 2.694) F1050 3 1.8887 0.0331 (1.6441, 2.1332) F1051 3 1.9179 0.153 1 (1.6733, 2.1625) F2001R 3 2.0597 0.0491 (1.8151, 2.3043) F2002 3 2.3967 0.1263 (2.1521, 2.6412) F2003 3 1.9778 0.1562 (1.7332, 2.2224) F2004 3 2.0627 0.1279 (1.8181, 2.3073) F2005 3 2.1266 0.0791 (1.8820, 2.3711) Table 13.8 F2006 3 2.2269 0.1537 (1.9823, 2.4715) F2008 3 2.1221 0.1578 (1.8775, 2.3666) F2009 3 2.004 0.270 ( 1.760, 2.249) F2011 3 2.282 0.245 ( 2.037, 2.526) F2012 3 2.075 0.210 ( 1.830, 2.319) F2014R 3 1.9889 0.1208 (1.7443, 2.2335) F2015 3 2.0398 0.1520 (1.7952, 2.2844) F2016 3 2.248 0.332 ( 2.003, 2.492) F2018 3 2.552 0.416 ( 2.307, 2.796) F2019 3 2.490 0.385 ( 2.246, 2.735) F2020 3 2.346 0.220 ( 2.101, 2.590) F2021 3 1.622 0.197 ( 1.378, 1.867) F2022 3 2.209 0.236 ( 1.964, 2.453) F2024R 3 2.223 0.287 ( 1.978, 2.467) F2025R 3 2.3432 0.0578 (2.0987, 2.5878) F2027R 3 1.918 0.186 ( 1.673, 2.163) F2028R 3 2.170 0.288 ( 1.925, 2.415) F2029R 3 2.2650 0.0621 (2.0204, 2.5095) F2030 3 2.3733 0.0585 (2.1287, 2.6179) F2031 3 2.108 0.175 ( 1.864, 2.353) F2032 3 2.1560 0.1358 (1.9114, 2.4006) F2033 3 1.699 0.173 ( 1.454, 1.943) F2034 3 1.9805 0.1499 (1.7359, 2.2250) F2035 3 1.7422 0.0863 (1.4977, 1.9868) F2036 3 2.2444 0.1423 (1.9999, 2.4890) F2037 3 2.3199 0.1639 (2.0754, 2.5645) F2038 3 1.7902 0.0784 (1.5457, 2.0348) F2039 3 2.2623 0.1069 (2.0177, 2.5069) F2040 3 1.9902 0.1530 (1.7456, 2.2348) F2041 3 2.1082 0.1335 (1.8636, 2.3528) F2042 3 1.8751 0.1026 (1.6305, 2.1196) I5440 3 2.309 0.665 ( 2.065, 2.554) I7067 3 2.1964 0.1014 (1.9518, 2.4409) I7127 3 2.2146 0.0877 (1.9700, 2.4592) I7826 3 2.263 0.176 ( 2.018, 2.508) I9339 3 2.0485 0.0332 (1.8039, 2.2931) I9340 3 2.3329 0.0452 (2.0884, 2.5775) Pooled StDev = 0.213838 Fisher Pairwise Comparisons Grouping Information Using the Fisher LSD Method and 95% Confidence C1 N Mean Grouping F2018 3 2.552 A F2019 3 2.490 A B F1049 3 2.450 A B C F2002 3 2.3967 A B C D F2030 3 2.3733 A B C D E F2020 3 2.346 A B C D E F F2025R 3 2.3432 A B C D E F F1012 3 2.340 A B C D E F I9340 3 2.3329 A B C D E F G F1048 3 2.328 A B C D E F G F2037 3 2.3199 A B C D E F G H I5440 3 2.309 A B C D E F G H F2011 3 2.282 A B C D E F G H F1027 3 2.2723 A B C D E F G H I F2029R 3 2.2650 A B C D E F G H I I7826 3 2.263 A B C D E F G H I J F2039 3 2.2623 A B C D E F G H I J F2016 3 2.248 A B C D E F G H I J F2036 3 2.2444 A B C D E F G H I J F2006 3 2.2269 A B C D E F G H I J K F2024R 3 2.223 A B C D E F G H I J K I7127 3 2.2146 A B C D E F G H I J K L F2022 3 2.209 A B C D E F G H I J K L I7067 3 2.1964 B C D E F G H I J K L F2028R 3 2.170 B C D E F G H I J K L M F2032 3 2.1560 B C D E F G H I J K L M F1029 3 2.1279 C D E F G H I J K L M N F1003R 3 2.1276 C D E F G H I J K L M N F2005 3 2.1266 C D E F G H I J K L M N F2008 3 2.1221 C D E F G H I J K L M N F1026R 3 2.121 C D E F G H I J K L M N E0028 3 2.1138 C D E F G H I J K L M N F2031 3 2.108 C D E F G H I J K L M N F 2041 3 2.1082 C D E F G H I J K L M N F1047 3 2.093 D E F G H I J K L M N F2012 3 2.075 D E F G H I J K L M N O F1032R 3 2.0670 D E F G H I J K L M N O F2004 3 2.0627 D E F G H I J K L M N O F2001R 3 2.059 7 D E F G H I J K L M N O I9339 3 2.0485 E F G H I J K L M N O F1014 3 2.041 E F G H I J K L M N O P F2015 3 2.0398 E F G H I J K L M N O P F2009 3 2.004 F G H I J K L M N O P F2040 3 1.9902 G H I J K L M N O P F2014R 3 1.9889 G H I J K L M N O P F2034 3 1.9805 H I J K L M N O P F2003 3 1.9778 H I J K L M N O P E1007R 3 1.9314 I J K L M N O P Q F2027R 3 1.918 J K L M N O P Q F1051 3 1.9179 J K L M N O P Q F1050 3 1.8887 K L M N O P Q F2042 3 1.8751 L M N O P Q E5011 3 1.846 M N O P Q F2038 3 1.7902 N O P Q F2035 3 1.7422 O P Q F2033 3 1.699 P Q F2021 3 1.622 Q Means that do not share a letter are significantly dif ferent. Table 13 .9. Raw Data for 2013 Free Phenolic Content One - way ANOVA: C2 versus C1 * ERROR * Cannot draw the interval plot. Interval plots are illegible with more than 45 intervals. * NOTE * Cannot draw the interval plot for the Fisher procedure. Interval plots for comparisons are illegible with more than 45 intervals. Method Null hypothesis All means are equal Alternative hypothesis At least one mean is different Equal variances were assumed for the analysis. Analysis of Variance Source DF Adj SS Adj MS F - Value P - Value C1 56 97.58 1.743 1.11 0.319 Error 114 179.33 1.573 Total 170 276.91 Model Summary S R - sq R - sq(adj) R - sq(pred) 1.25423 35.24% 3.43% 0.00% Means C1 N Mean StDev 95% CI E0028 3 3.270 1.078 (1.836, 4.705) E1007R 3 3.007 1.033 (1.573, 4.442) E5011 3 5.848 1.034 (4.413, 7.282) F1003R 3 2.516 1.286 (1.081, 3.950) F1012 3 3.354 1.7 03 (1.919, 4.788) F1014 3 5.436 1.009 (4.002, 6.871) F1026R 3 3.266 1.328 (1.832, 4.701) F1027 3 2.621 1.367 (1.187, 4.056) F1029 3 4.100 1.082 (2.666, 5.534) F1032R 3 3.513 1.201 (2.079, 4.948) F1047 3 2.569 1.170 (1.135, 4.00 4) F1048 3 3.122 1.631 (1.687, 4.556) F1049 3 2.176 1.321 (0.741, 3.610) F1050 3 3.297 1.129 (1.862, 4.731) F1051 3 2.333 1.099 (0.898, 3.767) F2001R 3 3.765 1.160 (2.330, 5.199) F2002 3 2.822 1.453 (1.387, 4.256) F2003 3 4.636 1.010 (3.201, 6.070) F2004 3 3.340 1.265 (1.905, 4.774) F2005 3 2.819 1.284 (1.384, 4.253) F2006 3 3.737 1.190 (2.303, 5.172) F2008 3 3.400 1.389 (1.966, 4.835) F2009 3 3.128 0.933 (1.693, 4.562) F2011 3 3.684 1.071 (2.249, 5.118) F2012 3 2.481 1.143 (1.046, 3.915) F2014R 3 3.584 1.053 (2.150, 5.019) F2015 3 3.265 1.243 (1.831, 4.700) F2016 3 2.741 1.439 (1.307, 4.176) F2018 3 2.47 1.75 ( 1.04, 3.91) F2019 3 2.400 1.51 3 (0.966, 3.834) F2020 3 2.291 1.481 (0.856, 3.725) F2021 3 3.874 0.701 (2.440, 5.309) F2022 3 3.652 1.275 (2.217, 5.086) F2024R 3 2.887 1.400 (1.452, 4.321) F2025R 3 4.561 1.420 (3.126, 5.995) F2027R 3 3.888 0.907 (2.454, 5.323 ) F2028R 3 2.910 1.382 (1.476, 4.345) F2029R 3 4.105 1.383 (2.670, 5.539) F2030 3 4.737 1.441 (3.302, 6.171) F2031 3 3.362 1.028 (1.927, 4.796) F2032 3 2.421 1.369 (0.986, 3.855) F2033 3 3.466 0.814 (2.032, 4.901) F2034 3 3.071 1.066 (1.636, 4.505) F2035 3 3.950 0.996 (2.516, 5.385) F2036 3 2.793 1.392 (1.359, 4.228) F2037 3 2.273 1.339 (0.839, 3.708) F2038 3 3.757 0.913 (2.323, 5.192) F2039 3 3.852 1.438 (2.417, 5.286) F2040 3 3.648 1.260 (2 .214, 5.083) F2041 3 2.912 1.292 (1.478, 4.347) F2042 3 2.785 0.971 (1.350, 4.219) I5440 3 3.584 1.239 (2.149, 5.018) I7067 3 2.499 1.308 (1.065, 3.934) I7127 3 3.712 1.377 (2.277, 5.146) I7826 3 3.372 1.324 (1.938, 4.807) I93 39 3 2.790 1.222 (1.355, 4.224) I9340 3 2.830 1.360 (1.395, 4.264) Pooled StDev = 1.25423 Fisher Pairwise Comparisons Grouping Information Using the Fisher LSD Method and 95% Confidence C1 N Mean Grouping E5011 3 5.848 A F1014 3 5.436 A B F2030 3 4.737 A B C F2003 3 4.636 A B C D F2025R 3 4.561 A B C D E F2029R 3 4.105 A B C D E F F1029 3 4.100 A B C D E F F2035 3 3.950 A B C D E F F2027R 3 3.888 A B C D E F F2021 3 3.874 A B C D E F F2039 3 3.852 A B C D E F F2001R 3 3.765 B C D E F F2038 3 3.757 B C D E F F2006 3 3.737 B C D E F I7127 3 3.712 B C D E F F2011 3 3.684 B C D E F F2022 3 3.652 B C D E F F2040 3 3.648 B C D E F F2014R 3 3.584 B C D E F I5440 3 3.584 B C D E F F1032R 3 3.513 B C D E F F2033 3 3.466 B C D E F F2008 3 3.400 C D E F I7826 3 3.372 C D E F F2031 3 3.362 C D E F F1012 3 3.354 C D E F F2004 3 3.340 C D E F F1050 3 3.297 C D E F E0028 3 3.270 C D E F F1026R 3 3.266 C D E F F2015 3 3.265 C D E F F2009 3 3.128 C D E F F1048 3 3.122 C D E F F2034 3 3.071 C D E F E1007R 3 3.007 C D E F F2041 3 2.912 C D E F F2028R 3 2.910 C D E F F2024R 3 2.887 C D E F I9340 3 2.830 C D E F F2002 3 2.822 C D E F F2005 3 2.819 C D E F F2036 3 2.793 C D E F I9339 3 2.790 C D E F F2042 3 2.785 C D E F F2016 3 2.741 C D E F F1027 3 2.621 D E F F1047 3 2.569 E F F1003R 3 2.516 F I7067 3 2.499 F F2012 3 2.481 F F2018 3 2.47 F F2032 3 2.421 F F2019 3 2.400 F F1051 3 2.333 F F2020 3 2.291 F F2037 3 2.273 F F1049 3 2.176 F Means that do not share a letter are significantly different. Table 13 .10. Raw Data for 2014 Total Phenolic Content One - way ANOVA: C2 versus C1 * NOTE * Cannot draw the interval plot for the Fisher procedure. Interval plots for comparisons are illegible with more than 45 intervals. Method Null hypothesis All means are equal Alternative hypothesis A t least one mean is different Equal variances were assumed for the analysis. Analysis of Variance Source DF Adj SS Adj MS F - Value P - Value C1 38 23.91 0.6292 2.77 0.000 Error 78 17.72 0.2271 Total 116 41.63 Model Summary S R - sq R - sq(adj) R - sq(pred) 0.476597 57.44% 36.70% 4.24% Means C1 N Mean StDev 95% CI E0028 3 4.290 0.466 ( 3.743, 4.838) E5011 3 5.297 0.631 ( 4.749, 5.845) F00 13R 3 4.505 0.356 ( 3.957, 5.053) F1026R 3 5.728 0.561 ( 5.180, 6.276) F1027 3 5.205 0.249 ( 4.658, 5.753) F1029 3 5.098 0.565 ( 4.550, 5.646) F1047 3 5.208 0.815 ( 4.660, 5.756) F1048 3 4.903 0.874 ( 4.356, 5. 451) F1049 3 5.022 0.893 ( 4.474, 5.569) F2002 3 5.786 0.630 ( 5.238, 6.334) F2003 3 5.1284 0.1626 (4.5806, 5.6762) F2005 3 4.7871 0.0466 (4.2393, 5.3349) F2008 3 4.627 0.675 ( 4.079, 5.175) F2009 3 5.4218 0.1161 (4.8 740, 5.9696) F2010 3 4.360 0.238 ( 3.812, 4.908) F2012 3 4.4727 0.1164 (3.9249, 5.0205) F2014R 3 5.173 0.233 ( 4.625, 5.721) F2015 3 5.717 0.193 ( 5.170, 6.265) F2016 3 5.479 0.624 ( 4.931, 6.027) F2018 3 4.430 0.790 ( 3.882, 4.978) F2019 3 5.141 0.256 ( 4.593, 5.689) F2020 3 5.242 0.430 ( 4.695, 5.790) F2021 3 5.1504 0.1158 (4.6026, 5.6982) F2022 3 5.702 0.717 ( 5.154, 6.250) F2024R 3 5.515 0.492 ( 4.967, 6.063) F2028R 3 5.159 0.233 ( 4.611, 5.707) F2029R 3 4.696 0.584 ( 4.148, 5.243) F2030 3 5.0888 0.0748 (4.5410, 5.6366) F2031 3 5.1282 0.0578 (4.5804, 5.6760) F2033 3 5.2777 0.0531 (4.7299, 5.8255) F2034 3 5.698 0 .592 ( 5.150, 6.246) F2037 3 3.996 0.223 ( 3.448, 4.544) F2038 3 5.123 0.460 ( 4.575, 5.671) F2039 3 5.0285 0.1370 (4.4807, 5.5763) F2042 3 5.522 0.427 ( 4.974, 6.070) I7826 3 5.681 0.351 ( 5.133, 6.229) x1 3 5 .976 0.185 ( 5.428, 6.524) x2 3 5.250 0.841 ( 4.702, 5.798) x3 3 4.9431 0.1466 (4.3953, 5.4909) Pooled StDev = 0.476597 Fisher Pairwise Comparisons Grouping Information Using the Fisher LSD Method and 95% Confidence C1 N Mean Grouping x1 3 5.976 A F2002 3 5.786 A B F1026R 3 5.728 A B F2015 3 5.717 A B C F2022 3 5.702 A B C F2034 3 5.698 A B C I7826 3 5.681 A B C F2042 3 5.522 A B C D F2024R 3 5.515 A B C D F2016 3 5.479 A B C D F2009 3 5.4218 A B C D E E5011 3 5.297 A B C D E F F2033 3 5.2777 A B C D E F G x2 3 5.250 A B C D E F G F2020 3 5.242 A B C D E F G H F1047 3 5.208 A B C D E F G H F1027 3 5.205 A B C D E F G H F2014R 3 5.1 73 B C D E F G H I F2028R 3 5.159 B C D E F G H I F2021 3 5.1504 B C D E F G H I F2019 3 5.141 B C D E F G H I F2003 3 5.1284 B C D E F G H I J F2031 3 5.1282 B C D E F G H I J F2038 3 5.123 B C D E F G H I J F1029 3 5.098 B C D E F G H I J F2030 3 5.0888 B C D E F G H I J F2039 3 5.0285 B C D E F G H I J K F1049 3 5.022 B C D E F G H I J K x3 3 4.9431 C D E F G H I J K F1048 3 4.903 D E F G H I J K F2005 3 4.7871 D E F G H I J K F2029R 3 4.696 E F G H I J K L F2008 3 4.627 F G H I J K L F0013R 3 4.505 G H I J K L F2012 3 4.4727 H I J K L F2018 3 4.430 I J K L F2010 3 4.360 J K L E0028 3 4.290 K L F2037 3 3.996 L Means that do not share a letter are significantly different. Table 13 .11. Raw Data for 2014 Bound Phenolic Content One - wa y ANOVA: C2 versus C1 * NOTE * Cannot draw the interval plot for the Fisher procedure. Interval plots for comparisons are illegible with more than 45 intervals. Method Null hypothesis All means are equal Alternative hypothesis At least one mean is different Equal variances were assumed for the analysis. Analysis of Variance Source DF Adj SS Adj MS F - Value P - Value C1 38 27.65 0.7277 1.89 0.009 Error 78 30.09 0.3858 Tot al 116 57.75 Model Summary S R - sq R - sq(adj) R - sq(pred) 0.621119 47.89% 22.50% 0.00% Means C1 N Mean StDev 95% CI E0028 3 3.028 0.737 ( 2.314, 3.742) E5011 3 3.732 0.638 ( 3.018, 4.446) F0013R 3 3.771 0.310 ( 3.057, 4.485) F1026R 3 4.4918 0.1086 ( 3.7779, 5.2057) F1027 3 3.7433 0.0733 ( 3.0294, 4.4572) F1029 3 3.757 0.556 ( 3.043, 4.471) F1047 3 4.648 1.325 ( 3.934, 5.362) F1048 3 4.339 1.073 ( 3.625, 5.053) F1049 3 3.481 1.173 ( 2.767, 4.195) F2002 3 4.391 0.652 ( 3.677, 5.105) F2003 3 4.107 0.460 ( 3.393, 4.821) F2005 3 3.7699 0.1050 ( 3.0560, 4.4838) F2008 3 3.613 1.0 13 ( 2.899, 4.327) F2009 3 4.304 0.375 ( 3.590, 5.018) F2010 3 2.8708 0.1362 ( 2.1568, 3.5847) F2012 3 3.440 0.390 ( 2.726, 4.154) F2014R 3 3.82214 0.00245 (3.10821, 4.53606) F2015 3 4.6072 0.1625 ( 3.8932, 5 .3211) F2016 3 3.945 0.757 ( 3.231, 4.658) F2018 3 3.767 1.062 ( 3.053, 4.481) F2019 3 3.5131 0.0777 ( 2.7992, 4.2270) F2020 3 4.563 0.597 ( 3.849, 5.277) F2021 3 3.691 0.489 ( 2.977, 4.405) F2022 3 4.656 0.740 ( 3.942, 5.369) F2024R 3 4.424 0.813 ( 3.710, 5.138) F2028R 3 3.680 0.202 ( 2.966, 4.394) F2029R 3 3.106 0.739 ( 2.393, 3.820) F2030 3 3.965 0.407 ( 3.252, 4.679) F2031 3 3.8426 0.0590 ( 3.1287, 4.5565) F2033 3 4.113 0.330 ( 3.399, 4.827) F2034 3 4.136 0.827 ( 3.422, 4.850) F2037 3 3.01863 0.00262 (2.30470, 3.73255) F2038 3 3.53641 0.00875 (2.82248, 4.25033) F2039 3 4.1 90 0.380 ( 3.476, 4.904) F2042 3 4.537 0.333 ( 3.823, 5.251) I7826 3 4.161 0.779 ( 3.447, 4.875) x1 3 4.911 0.343 ( 4.197, 5.625) x2 3 3.731 1.026 ( 3.018, 4.445) x3 3 3.968 0.536 ( 3.254, 4.682) Pooled StDev = 0.621119 Fisher Pairwise Comparisons Grouping Information Using the Fisher LSD Method and 95% Confidence C1 N Mean Grouping x1 3 4.911 A F2022 3 4.656 A B F1047 3 4.648 A B F2015 3 4. 6072 A B C F2020 3 4.563 A B C F2042 3 4.537 A B C D F1026R 3 4.4918 A B C D E F2024R 3 4.424 A B C D E F F2002 3 4.391 A B C D E F F1048 3 4.339 A B C D E F F2009 3 4.304 A B C D E F F2039 3 4.190 A B C D E F I7826 3 4.161 A B C D E F F2034 3 4.136 A B C D E F F2033 3 4.113 A B C D E F G F2003 3 4.107 A B C D E F G x3 3 3.968 A B C D E F G H F2030 3 3.965 A B C D E F G H F2016 3 3.945 A B C D E F G H F2031 3 3.84 26 B C D E F G H I F2014R 3 3.82214 B C D E F G H I F0013R 3 3.771 B C D E F G H I F2005 3 3.7699 B C D E F G H I F2018 3 3.767 B C D E F G H I F1029 3 3.757 B C D E F G H I F1027 3 3.7433 B C D E F G H I E5011 3 3.732 B C D E F G H I x2 3 3.731 B C D E F G H I F2021 3 3.691 B C D E F G H I F2028R 3 3.680 B C D E F G H I F2008 3 3.613 C D E F G H I F2038 3 3.53641 D E F G H I F2019 3 3.5131 E F G H I F1049 3 3.481 F G H I F2012 3 3.440 F G H I F2029R 3 3.106 G H I E0028 3 3.028 H I F2037 3 3.01863 H I F2010 3 2.8708 I M eans that do not share a letter are significantly different. Table 13 .12. Raw Data for 2014 Free Phenolic Content Interval Plot of C2 vs C1 One - way ANOVA: C2 versus C1 * NOTE * Cannot draw the interval plot for the Fisher procedure. Interval plots for comparisons are illegible with more than 45 intervals. Method Null hypothesis All means are equal Alternative hypothesis At least one mean is different Equal variances were assumed for the analysis. Analysis of Variance Source DF Adj SS Adj MS F - Value P - Value C1 38 5.125 0.13486 2.24 0.001 Error 78 4.695 0.06020 Total 116 9.820 Model Summary S R - sq R - sq(adj) R - sq(pred) 0.245349 52.19% 2 8.89% 0.00% Means C1 N Mean StDev 95% CI E0028 3 1.4495 0.0292 ( 1.1675, 1.7315) E5011 3 1.3476 0.0321 ( 1.0656, 1.6296) F0013R 3 0.8457 0.0402 ( 0.5637, 1.1277) F1026R 3 1.373 0.345 ( 1.091, 1. 655) F1027 3 1.442 0.397 ( 1.160, 1.724) F1029 3 1.1249 0.0629 ( 0.8429, 1.4069) F1047 3 1.061 0.439 ( 0.779, 1.343) F1048 3 0.980 0.247 ( 0.698, 1.262) F1049 3 1.1686 0.0193 ( 0.8866, 1.4506) F2002 3 1.3404 0.0211 ( 1.0583, 1.6224) F2003 3 1.186 0.335 ( 0.904, 1.468) F2005 3 1.0475 0.0443 ( 0.7655, 1.3295) F2008 3 1.228 0.277 ( 0.946, 1.510) F2009 3 1.077 0.312 ( 0.795, 1.359) F2010 3 1.4497 0.1201 ( 1.1677, 1.7317) F2012 3 1.079 0.323 ( 0.797, 1.361) F2014R 3 1.298 0.348 ( 1.016, 1.580) F2015 3 1.1298 0.0632 ( 0.8478, 1.4118) F2016 3 1.1160 0.1362 ( 0.8340, 1.3980) F2018 3 1.12308 0 .00543 (0.84107, 1.40508) F2019 3 1.650 0.323 ( 1.368, 1.932) F2020 3 0.9182 0.1028 ( 0.6362, 1.2002) F2021 3 1.508 0.415 ( 1.226, 1.790) F2022 3 0.8330 0.0767 ( 0.5510, 1.1150) F2024R 3 1.192 0.269 ( 0.910 , 1.474) F2028R 3 1.3607 0.1160 ( 1.0787, 1.6427) F2029R 3 1.2426 0.1031 ( 0.9606, 1.5246) F2030 3 1.241 0.278 ( 0.959, 1.523) F2031 3 1.3360 0.0542 ( 1.0540, 1.6180) F2033 3 1.254 0.218 ( 0.972, 1.536) F2034 3 1.3233 0.0501 ( 1.0413, 1.6053) F2037 3 0.976 0.258 ( 0.694, 1.258) F2038 3 1.589 0.468 ( 1.307, 1.871) F2039 3 0.9818 0.1656 ( 0.6998, 1.2638) F2042 3 0.9745 0.1558 ( 0.6925, 1.2565) I7826 3 1.744 0.254 ( 1.462, 2.026) x1 3 1.188 0.511 ( 0.906, 1.470) x2 3 1.3340 0.0578 ( 1.0520, 1.6160) x3 3 1.180 0.270 ( 0.898, 1.462) Pooled StDev = 0.245349 Fisher Pairwise Comparisons Grouping Information Using the Fisher LSD Method and 95% Confidence C1 N Mean Grouping I7826 3 1.744 A F2019 3 1.650 A B F2038 3 1.589 A B C F2021 3 1.508 A B C D F2010 3 1.4497 A B C D E E0028 3 1.4495 A B C D E F1027 3 1.442 A B C D E F F1026R 3 1.373 A B C D E F G F2028R 3 1.3607 A B C D E F G E5011 3 1.3476 A B C D E F G F2002 3 1.3404 B C D E F G F2031 3 1.3360 B C D E F G x2 3 1.3340 B C D E F G F2034 3 1.3233 B C D E F G F2014R 3 1.298 B C D E F G H F2033 3 1.254 B C D E F G H F2029R 3 1.2426 C D E F G H I F2030 3 1.241 C D E F G H I F2008 3 1.228 C D E F G H I J F2024R 3 1.192 C D E F G H I J x1 3 1.188 D E F G H I J F2003 3 1.186 D E F G H I J x3 3 1.180 D E F G H I J F1049 3 1.1686 D E F G H I J F2015 3 1.1298 D E F G H I J F1029 3 1.1249 D E F G H I J F2018 3 1.12308 D E F G H I J F2016 3 1.1160 D E F G H I J F2012 3 1.079 E F G H I J F2009 3 1.077 E F G H I J F1047 3 1.061 E F G H I J F2005 3 1.0475 F G H I J F2039 3 0.9818 G H I J F1048 3 0.980 G H I J F2037 3 0.976 G H I J F2042 3 0.9745 G H I J F2020 3 0.9182 H I J F0013R 3 0.8457 I J F2022 3 0.8330 J Means that do not share a letter are significantly different. A PPENDIX D . Colorimetric Analysis Raw Data Table 14 .1. Phenolic Acid Contents 1 of Selected 2013 MSU Wheat Breeding Varieties Variety Number Variety Name Total Phenolic Content (mg GAE/g) Bound Phenolic Content (mg GAE/g) Free Phenolic Content (mg GAE/g) 1 F1014 4 . 2 ± 0.2 2 . 4 ± 0.4 2.2 ± 0.2 2 F1026R 5.7 ± 0.2 2.5 ± 0.0 2.0 ± 0.2 3 F1012 4.8 ± 0.4 2.6 ± 0.7 2.2 ± 0.3 4 F1003R 4.0 ± 0.0 1.9 ± 0.2 2.1 ± 0.2 5 F1029 3.8 ± 0.2 3.4 ± 0.2 2.1 ± 0.0 6 F1047 3.7 ± 0.1 1.8 ± 0.4 2.0 ± 0.4 7 F1032R 5.1 ± 0.0 2.8 ± 0.0 2.1 ± 0.0 8 F1027 4.4 ± 0.1 1.9 ± 0.1 2.2 ± 0.1 9 F1049 3.8 ± 0.3 1.5 ± 0.1 2.5 ± 0.4 10 F1051 3.8 ± 0.1 1.6 ± 0.1 1.9 ± 0.2 11 F1050 4.5 ± 0.0 2.7 ± 0.1 1.9 ± 0.0 12 F1048 4.9 ± 0.3 2.6 ± 0.8 2.1 ± 0.4 13 F2031 4.8 ± 0.3 2.7 ± 0.6 2.2 ± 0.2 14 F2033 4.6 ± 0.3 2.9 ± 0.3 1.8 ± 0.0 15 F2034 4.2 ± 0.1 2.3 ± 0.3 2.0 ± 0.2 16 F2032 3.9 ± 0.2 1.8 ± 0.3 2.1 ± 0.1 17 F2037 3.6 ± 0.3 1.4 ± 0.4 2.2 ± 0.1 18 F2035 4.8 ± 0.1 3.4 ± 0.0 1.8 ± 0.1 19 F2041 4.4 ± 0.1 2.3 ± 0.3 2.1 ± 0.2 20 F2036 4.3 ± 0.2 2.1 ± 0.1 2.2 ± 0.1 21 F2038 5.3 ± 0.2 3.2 ± 0.3 1.8 ± 0.0 22 F2040 4.9 ± 0.1 3.1 ± 0.3 1.9 ± 0.2 23 F2042 4.3 ± 0.2 2.1 ± 0.3 1.9 ± 0.1 24 F2039 5.4 ± 0.2 3.1 ± 0.4 2.2 ± 0.1 25 F2001R 5.0 ± 0.1 3.1 ± 0.1 2.0 ± 0.0 26 F2006 5.0 ± 0.1 3.0 ± 0.4 2.2 ± 0.2 27 F2005 5.1 ± 0.0 2.1 ± 0.2 2.1 ± 0.1 28 F2015 4.7 ± 0.2 2.6 ± 0.0 2.0 ± 0.2 29 F2009 4.9 ± 0.4 2.4 ± 0.3 2.2 ± 0.1 30 F2012 4.6 ± 0.2 1.9 ± 0.1 2.1 ± 0.2 31 F2008 4.3 ± 0.3 2.8 ± 0.4 2.0 ± 0.0 32 F2011 5.0 ± 0.4 2.9 ± 0.5 2.4 ± 0.1 33 F2014R 5.3 ± 0.2 2.9 ± 0.1 2.1 ± 0.1 34 F2003 6.0 ± 0.2 3.9 ± 0.3 2.1 ± 0.0 35 F2004 4.9 ± 0.2 2.6 ± 0.1 2.0 ± 0.1 36 F2002 4.8 ± 0.0 2.1 ± 0.2 2.3 ± 0.1 37 F2028R 4.3 ± 0.2 2.3 ± 0.2 2.0 ± 0.1 38 F2020 3.8 ± 0.2 1.6 ± 0.2 2.2 ± 0.0 39 F2024R 4.8 ± 0.0 2.4 ± 0.3 2.1 ± 0.2 40 F2018 4.2 ± 0.3 1.9 ± 0.6 2.3 ± 0.2 41 F2019 3.9 ± 0.2 1.7 ± 0.4 2.3 ± 0.2 42 F2016 4.3 ± 0.1 2.2 ± 0.5 2.2 ± 0.4 43 F2022 5.3 ± 0.0 3.0 ± 0.2 2.1 ± 0.2 44 F2025R 5.9 ± 0.1 3.8 ± 0.1 2.3 ± 0.0 45 F2021 5.1 ± 0.5 3.5 ± 0.5 1.7 ± 0.1 46 F2030 6.0 ± 0.1 4.0 ± 0.2 2.3 ± 0.0 47 F2029R 5.6 ± 0.2 3.4 ± 0.2 2.2 ± 0.0 48 F2027R 4.8 ± 0.3 3.1 ± 0.7 2.0 ± 0.2 49 Aubrey 4.9 ± 0.0 2.7 ± 0.2 2.3 ± 0.2 50 Hopewell 4.7 ± 0.2 3.0 ± 0.2 2.2 ± 0.0 51 Ambassador 5.2 ± 0.3 2.5 ± 0.5 2.2 ± 0.0 52 Jupiter 6.8 ± 0.1 5.3 ± 0.3 1.8 ± 0.2 53 Red Ruby 3.9 ± 0.2 2.4 ± 0.0 2.0 ± 0.1 54 VA09W - 188WS 4.1 ± 0.1 2.1 ± 0.1 2.0 ± 0.0 55 Cayuga 5.1 ± 0.2 2.5 ± 0.7 2.3 ± 0.4 56 VA09W - 192WS 4.2 ± 0.0 2.1 ± 0.1 2.3 ± 0.1 57 Caledonia 4.1 ± 0.1 1.7 ± 0.2 2.2 ± 0.1 1 Results are expressed as milligrams of gallic acid equivalent per gram of sample, with the data presented as means ± standard deviation (n=3). Table 14 .2. Phenolic Acid Contents 1 of Selected 2014 MSU Wheat Breeding Varieties Variety Number Variety Name Total Phenolic Content (mg GAE/g) Bound Phenolic Content (mg GAE/g) Free Phenolic Content (mg GAE/g) 2 F1026R 5.8 ± 0.5 4.4 ± 0.0 1.4 ± 0.6 5 F1029 5.1 ± 0.4 4.0 ± 0.5 1.1 ± 0.1 6 F1047 5.2 ± 0.5 4.3 ± 1.6 0.9 ± 0.5 8 F1027 5.2 ± 0.4 3.8 ± 0.1 1.4 ± 0.3 9 F1049 5.0 ± 0.3 3.8 ± 1.4 1.2 ± 0.0 12 F1048 4.9 ± 0.4 4.0 ± 1.2 1.0 ± 0.3 13 F2031 5.2 ± 0.0 3.8 ± 0.1 1.3 ± 0.1 14 F2033 5.2 ± 0.1 4.0 ± 0.4 1.2 ± 0.2 15 F2034 5.7 ± 0.5 4.4 ± 1.0 1.3 ± 0.1 17 F2037 4.0 ± 0.4 3.0 ± 0.0 1.0 ± 0.4 21 F2038 5.1 ± 0.7 3.5 ± 0.0 1.6 ± 0.7 23 F2042 5.5 ± 0.5 4.5 ± 0.5 1.0 ± 0.2 24 F2039 5.1 ± 0.3 4.1 ± 0.5 1.0 ± 0.2 27 F2005 4.8 ± 0.1 3.7 ± 0.1 1.0 ± 0.1 28 F2015 5.7 ± 0.3 4.6 ± 0.2 1.1 ± 0.1 29 F2009 5.5 ± 0.0 4.4 ± 0.5 1.1 ± 0.4 30 F2012 4.4 ± 0.0 3.3 ± 0.5 1.1 ± 0.5 31 F2008 4.5 ± 0.6 3.3 ± 1.2 1.2 ± 0.4 33 F2014R 5.1 ± 0.3 3.8 ± 0.0 1.3 ± 0.3 34 F2003 5.1 ± 0.2 3.9 ± 0.5 1.2 ± 0.3 36 F2002 5.9 ± 0.4 4.6 ± 0.8 1.3 ± 0.0 37 F2028R 5.0 ± 0.2 3.6 ± 0.2 1.4 ± 0.0 38 F2020 5.3 ± 0.6 4.4 ± 0.7 0.9 ± 0.1 39 F2024R 5.4 ± 0.6 4.2 ± 1.0 1.2 ± 0.4 40 F2018 4.7 ± 0.4 3.5 ± 1.4 1.1 ± 0.0 41 F2019 5.1 ± 0.4 3.5 ± 0.1 1.7 ± 0.5 42 F2016 5.3 ± 0.7 4.2 ± 0.9 1.1 ± 0.2 43 F2022 5.7 ± 0.5 4.9 ± 0.9 0.8 ± 0.1 45 F2021 5.1 ± 0.0 3.6 ± 0.6 1.5 ± 0.6 46 F2030 5.1 ± 0.1 3.8 ± 0.5 1.2 ± 0.4 47 F2029R 4.6 ± 0.4 3.3 ± 0.9 1.2 ± 0.1 49 Aubrey 5.7 ± 0.2 3.9 ± 1.0 1.7 ± 0.3 51 Ambassador 4.3 ± 0.7 2.8 ± 0.9 1.4 ± 0.0 52 Jupiter 5.3 ± 0.7 3.9 ± 0.8 1.3 ± 0.0 58 MSU Line F2010 4.4 ± 0.3 2.9 ± 0.2 1.4 ± 0.2 59 Unnamed 1 6.0 ± 0.3 4.8 ± 0.4 1.2 ± 0.6 60 Unnamed 2 5.3 ± 0.4 4.0 ± 1.3 1.3 ± 0.3 61 Unnamed 3 4.9 ± 0.2 3.9 ± 0.7 1.1 ± 0.5 62 F0013R 4.5 ± 0.3 3.6 ± 0.3 0.9 ± 0.0 1 Results are expressed as milligrams of gallic acid equivalent per gram of sample, with the data presented as means ± standard deviation (n=3). Table 14 .3. Total Phenolic Acid Contents 1 of 2013 Wheat Fractions Variety Number Variety Name Bran TPC (mg GAE/g) Shorts TPC (mg GAE/g) Flour TPC (mg GAE/g) 1 F1014 8.2 ± 0.5 7.5 ± 0.5 1.9 ± 0.7 2 F1026R 7.9 ± 0.2 6.0 ± 0.1 2.2 ± 0.6 3 F1012 7.3 ± 0.8 6.8 ± 0.4 2.3 ± 0.1 4 F1003R 8.5 ± 0.5 7.3 ± 0.1 1.8 ± 0.5 5 F1029 7.8 ± 1.1 8.0 ± 0.4 2.9 ± 0.1 6 F1047 8.4 ± 0.3 7.7 ± 0.4 2.5 ± 0.3 7 F1032R 9.1 ± 1.1 8.3 ± 0.3 2.2 ± 0.1 8 F1027 9.4 ± 0.3 5.6 ± 0.3 1.7 ± 0.3 9 F1049 8.6 ± 0.5 5.5 ± 0.9 2.0 ± 0.7 10 F1051 9.4 ± 0.9 9.5 ± 0.0 2.0 ± 0.3 11 F1050 8.2 ± 0.1 8.0 ± 0.4 2.7 ± 0.0 12 F1048 7.5 ± 0.5 7.4 ± 0.3 2.3 ± 0.2 13 F2031 8.6 ± 0.2 9.4 ± 0.3 1.5 ± 0.3 14 F2033 8.4 ± 0.0 8.9 ± 0.7 2.1 ± 0.2 15 F2034 10.4 ± 0.9 10.1 ± 0.5 2.4 ± 0.7 16 F2032 8.5 ± 0.3 7.0 ± 0.5 2.4 ± 0.7 17 F2037 9.0 ± 1.1 5.9 ± 0.5 2.6 ± 0.8 18 F2035 10.3 ± 0.0 5.0 ± 0.5 2.1 ± 0.9 19 F2041 9.4 ± 0.7 6.7 ± 0.1 1.6 ± 0.9 20 F2036 9.4 ± 0.0 8.0 ± 0.4 2.3 ± 0.4 21 F2038 7.9 ± 1.1 8.0 ± 1.0 1.3 ± 0.3 22 F2040 10.7 ± 0.7 7.0 ± 0.3 1.7 ± 0.4 23 F2042 9.4 ± 0.7 8.7 ± 0.7 2.1 ± 0.5 24 F2039 8.1 ± 0.1 7.7 ± 1.3 2.0 ± 0.6 25 F2001R 8.9 ± 0.5 8.0 ± 0.4 2.3 ± 0.8 26 F2006 8.1 ± 0.4 7.7 ± 1.2 2.1 ± 0.3 27 F2005 8.6 ± 1.0 8.6 ± 0.9 1.8 ± 0.7 28 F2015 9.2 ± 0.3 8.9 ± 1.6 2.4 ± 0.3 29 F2009 9.4 ± 0.6 8.8 ± 0.3 2.1 ± 0.7 30 F2012 8.8 ± 0.1 6.8 ± 1.2 2.1 ± 0.8 31 F2008 8.8 ± 0.2 7.3 ± 0.6 1.9 ± 0.5 32 F2011 13.0 ± 0.7 10.7 ± 0.4 3.3 ± 0.1 33 F2014R 8.6 ± 0.7 8.3 ± 0.5 2.0 ± 0.3 34 F2003 9.4 ± 0.4 8.5 ± 0.5 2.1 ± 0.3 35 F2004 7.6 ± 0.1 7.5 ± 0.4 1.7 ± 0.2 36 F2002 8.6 ± 0.4 7.9 ± 0.3 2.5 ± 0.6 37 F2028R 8.5 ± 0.5 8.4 ± 0.5 1.3 ± 0.6 38 F2020 6.9 ± 0.1 8.9 ± 1.0 1.4 ± 0.5 39 F2024R 10.2 ± 0.2 9.0 ± 0.8 1.8 ± 1.0 40 F2018 9.3 ± 0.3 9.1 ± 0.2 1.5 ± 0.9 41 F2019 9.0 ± 0.2 8.2 ± 0.5 2.1 ± 0.7 42 F2016 6.6 ± 0.1 8.7 ± 0.3 2.7 ± 0.7 43 F2022 11.3 ± 0.3 7.8 ± 0.2 2.9 ± 0.7 Table 14.3 44 F2025R 9.3 ± 0.7 7.6 ± 0.1 1.5 ± 0.7 45 F2021 10.4 ± 1.4 9.4 ± 0.3 2.9 ± 0.9 46 F2030 11.9 ± 0.3 10.5 ± 0.2 2.4 ± 0.8 47 F2029R 9.7 ± 0.9 8.5 ± 0.2 1.8 ± 0.9 48 F2027R 9.6 ± 0.1 7.7 ± 0.3 2.1 ± 0.7 49 Aubrey 9.6 ± 0.3 8.1 ± 0.7 2.0 ± 0.6 50 Hopewell 9.4 ± 0.2 7.6 ± 0.2 2.7 ± 0.9 51 Ambassador 8.1 ± 0.2 6.1 ± 0.4 1.6 ± 0.8 52 Jupiter 9.1 ± 0.2 8.9 ± 0.5 2.4 ± 0.7 53 Red Ruby 8.4 ± 0.2 5.6 ± 0.5 1.3 ± 0.9 54 VA09W - 188WS 8.4 ± 0.3 7.6 ± 0.1 1.5 ± 0.8 55 Cayuga 8.3 ± 0.1 8.7 ± 0.2 2.1 ± 0.5 56 VA09W - 192WS 8.2 ± 0.2 8.4 ± 0.4 2.4 ± 0.7 57 Caledonia 7.8 ± 0.2 8.3 ± 0.4 1.9 ± 0.7 1 Results are expressed as milligrams of gallic acid equivalent per gram of sample, with the data presented as means ± standard deviation (n=3). Table 14 .4. Total Phenolic Acid Contents 1 of 2014 Wheat Fractions Variety Number Variety Name Bran TPC (mg GAE/g) Shorts TPC (mg GAE/g) Flour TPC (mg GAE/g) 2 F1026R 9.7 ± 0.4 7.9 ± 0.5 2.0 ± 0.3 5 F1029 10.4 ± 0.5 9.7 ± 1.1 2.3 ± 0.3 6 F1047 10.2 ± 0.4 8.1 ± 0.8 1.9 ± 0.2 8 F1027 9.1 ± 0.2 7.9 ± 0.7 1.9 ± 0.5 9 F1049 8.8 ± 0.8 7.3 ± 1.6 3.0 ± 0.3 12 F1048 11.5 ± 0.6 8.4 ± 0.8 2.6 ± 0.3 13 F2031 10.8 ± 0.1 8.7 ± 0.8 2.6 ± 0.2 14 F2033 9.8 ± 0.2 8.5 ± 1.0 2.7 ± 1.7 15 F2034 11.3 ± 0.4 8.0 ± 1.2 2.9 ± 1.1 17 F2037 9.1 ± 0.8 6.4 ± 1.2 2.2 ± 0.5 21 F2038 9.4 ± 0.5 9.7 ± 0.6 1.5 ± 0.2 23 F2042 9.8 ± 0.4 8.6 ± 1.0 2.5 ± 0.3 24 F2039 9.5 ± 1.0 10.2 ± 2.3 2.3 ± 0.0 27 F2005 9.7 ± 0.7 7.7 ± 0.4 2.1 ± 0.1 28 F2015 10.5 ± 0.0 8.9 ± 0.6 1.9 ± 0.5 29 F2009 9.5 ± 0.4 7.1 ± 0.2 2.0 ± 0.0 30 F2012 8.9 ± 0.3 7.7 ± 0.8 1.8 ± 0.8 31 F2008 9.5 ± 0.1 8.6 ± 0.8 2.2 ± 0.2 33 F2014R 9.4 ± 0.2 8.0 ± 1.4 2.7 ± 0.4 34 F2003 9.8 ± 0.3 7.5 ± 1.3 3.2 ± 0.3 36 F2002 10.4 ± 0.0 9.2 ± 0.8 2.3 ± 0.3 37 F2028R 10.0 ± 0.3 9.4 ± 0.1 2.4 ± 0.1 38 F2020 12.4 ± 0.9 8.6 ± 2.2 2.2 ± 0.5 39 F2024R 10.0 ± 0.5 8.7 ± 0.6 2.5 ± 0.2 40 F2018 10.4 ± 1.4 8.5 ± 1.6 2.2 ± 0.7 41 F2019 10.0 ± 0.5 8.1 ± 1.6 2.4 ± 0.3 42 F2016 10.1 ± 0.7 8.6 ± 0.8 2.1 ± 0.0 43 F2022 9.9 ± 0.2 8.6 ± 0.1 2.1 ± 0.9 45 F2021 9.7 ± 0.6 9.6 ± 0.3 2.5 ± 0.1 46 F2030 11.9 ± 0.3 8.2 ± 1.3 2.0 ± 0.2 47 F2029R 10.1 ± 0.6 9.3 ± 0.5 2.7 ± 0.6 49 Aubrey 9.5 ± 1.1 7.8 ± 0.2 2.6 ± 0.1 51 Ambassador 13.0 ± 1.5 8.0 ± 0.1 1.9 ± 0.6 52 Jupiter 11.1 ± 0.1 9.3 ± 0.3 2.1 ± 0.1 58 MSU Line F2010 9.7 ± 0.0 7.8 ± 0.0 1.7 ± 0.3 59 Unnamed 1 10.1 ± 0.7 8.7 ± 0.1 2.1 ± 0.2 60 Unnamed 2 10.5 ± 0.1 7.7 ± 0.3 3.1 ± 0.2 61 Unnamed 3 9.5 ± 0.2 7.5 ± 0.3 2.2 ± 0.2 62 F0013R 10.1 ± 0.4 9.1 ± 0.6 2.2 ± 0.0 1 Results are expressed as milligrams of gallic acid equivalent per gram of sample, with the data presented as means ± standard deviation (n=3). 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