Phenolic Compounds in Grains of Australian-Grown Sorghums: Quantitative Analyses including Impacts of Malting and Effects on Broiler Nutrition

Abstract

Grain sorghum (Sorghum bicolor) is one of the most important food and animal feed crops in arid and semi-arid regions of the world. Sorghum grain is rich in starch and contains many functional health-promoting compounds including B-group vitamins, vitamin E and a broad range of phenolics. There is increasing interest internationally to extend the commercial use of sorghum grain as an affordable but nutritious source of animal feed and as a functional food. The utilisation of energy by broiler chickens offered sorghum-based diets seems inadequate due to various factors such as kafirin, phytate and phenolic compounds, which may limit energy utilisation. The increased use of sorghum as a human food might be achieved partly through understanding the effects of various forms of grain processing. Malting is a biotechnological process that involves soaking, germination and kilning of the grain. For sorghum, malting has been identified as an effective process to increase the bioavailability of the macro- and micronutrients for sorghum based-foods but many questions concerning the biochemical effects of malting remain unanswered. There were two primary objectives for the research presented in this thesis: (1) To assess whether the abundance of phenolic compounds of a range of different classes influences energy utilisation in broiler chickens fed sorghum-based diets (Chapter 3), and (2) To evaluate the impact of malting on the abundance of several classes of sorghum phenolics (Chapter 4 to 6). A substantial collection of Australian-grown sorghum grains were analyzed for total phenolics, flavan-4-ols, antioxidant activity (DPPH; 2,2-diphenyl-1-picrylhydrazyl and ABTS; 2,2’-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid)), total flavonoids and total anthocyanins. Separation, identification and quantification of 3-deoxyanthocyanins, flavones, flavanones, as well as free, conjugated and bound phenolic acids, were also performed using high performance liquid chromatography (HPLC) and liquid chromatography coupled with mass spectrometry (LC-MS). Objective (1) was achieved through collaboration with experts in broiler chicken digestive physiology. A pigmented testa was found to be absent in all the Australian-grown sorghum cultivars examined (16 and 8 cultivars for Objectives (1) and (2), respectively), indicating that they were type I sorghums, which (by definition) lack condensed tannin. Analyses and assessments of other polyphenolic compounds, including flavan-4-ols and phenolic acids, appear to show that these have negative influences on broiler growth and meat production. The total phenolic content (TPC), in vitro antioxidant activity (DPPH and ABTS) and level of flavan-4-ols in the Australian sorghum grains ranged from 3.00 to 4.68 mg GAE/g, 8.50 to 14.47 mol TE/g, 18.81 to 33.73 umol TE/g and 0.84 to 7.98 abs/ml/g, respectively, where /g indicates per gram of grain on a dry matter basis in each case, as used for all experiments reported in this thesis. The total anthocyanin and total flavonoid level ranged from 1.30 to 11.55 abs/ml/g and 0.65 to 1.80 mg CE/g among the Australian sorghums tested. Levels of 3-deoxyanthocyanin ranged from 10.07 to 63.80 μg/g. The flavones detected in sorghum grain were apigenin (pale yellow) and luteolin (yellow). The flavone level ranged from 5.76 to 17.55 μg/g. The total content of flavanones (eriodictyol and naringenin) ranged from 29.39 to 189.62 μg/g, of which the eriodictyol content varied from 28.1 to 55.5% of total flavanones. Naringenin represented all of the flavanone content in one of the cultivars tested (cv. Block). Ferulic, caffeic, p-coumaric, syringic, vanillic, p-hydroxybenzoic and sinapic acids were detected in the grains of all sorghum cultivars tested. The total abundance of free phenolic acids ranged from 9.20 to 38.55 g/g. The most abundant of the free phenolic acids was caffeic acid, which varied from 1.47 to 18.35 g/g. Levels of total soluble conjugated phenolic acids in the sorghum grains varied from 44.50 to 96.76 g/g. Bound phenolic acids were the most abundant form of phenolic acids, with 228.57 to 580.33 g/g. Ferulic and p-coumaric acids were the predominant conjugated and bound phenolic acids among all the sorghum cultivars examined. In determining the effects of malting (soaking, germination and kilning) on the abundance of the grain phenolic compounds, eight different sorghum hybrids harvested from three different regions in eastern Australia in 2011 were analyzed. All the sorghum grains were tannin free based on both a bleach test and a vanillin-HCl assay. Malting decreased the total phenolic content and in vitro antioxidant activity in all samples. The grand means for total phenolics among all the sorghum cultivars were 2.77 and 2.48 mg GAE/g for raw and malted grain, respectively. For flavan-4-ols, the grand means for raw and malted sorghum grains were 2.98 and 2.23 abs/ml/g, respectively. On average, malting reduced the content of flavan-4-ols by 25%. Total anthocyanin levels more than doubled upon malting the sorghum whereas the total flavonoid level decreased by 12%. Luteolinidin, apigeninidin, 5-methoxy-luteolinidin and 7-methoxy-apigeninidin, which were detected as major 3-deoxyanthocyanins in all raw sorghum grains, were all found to increase in abundance upon malting. The content of luteolinindin and apigeninidin increased dramatically from 4.28 to 43.93 μg/g and from 4.43 to 37.89 μg/g, respectively. Overall, the level of flavones (apigenin and luteolin) increased by 68% after sorghum malting, whereas the level of flavanones decreased by 50%. The grand means for total free phenolic acids in raw and malted grains among the eight sorghum cultivars were 29.65 and 22.33 g/g, respectively. Reflecting the result for the distinct set of cultivars analysed above, caffeic acid was the most abundant free phenolic acid. The grand means for conjugated phenolic acids in the raw and malted sorghums were 74.88 and 73.46 g/g, respectively. While these values are very similar, malting did significantly affect the abundance of all conjugated individual phenolic acids. The grand means for bound phenolic acids in the raw and malted sorghums were 391.81 and 378.06 g/g, respectively. There was statistically significant change upon malting in the abundance of ferulic acid as major bound phenolic acid (344.71 and 333.02 g/g in raw and malted grain, respectively). The data obtained in this study on phenolic compounds in sorghum grain should be valuable for sorghum breeders in the selection of lines for specific end uses, food scientists developing sorghum-based products, broiler meat producers, and in the nutraceutical industry.