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Home , Dietary fiber, Phytosterol

Pearling and to Produce -Rich Fractions Anna-Maija Lampia,*, Robert A. Moreaub, Vieno Piironena, and Kevin B. Hicksb aDepartment of Applied Chemistry and Microbiology, University of Helsinki, Finland, and bUSDA, ARS, Eastern Regional Research Center, Wyndmoor, Pennsylvania 19038,

ABSTRACT: Because of the positive health effects of phyto- in the kernels and are more concentrated in the outer layers , phytosterol-enriched and foods containing than in the -rich endosperm (6,7). During the milling of elevated levels of natural are being developed. some grains, pearling is a traditional way of gradually remov- Phytosterol contents in are moderate, whereas their lev- ing the hull, pericarp, and other outer layers of the kernels and els in the outer layers of the kernels are higher. The phytosterols germ as pearling fines to produce pearled grains. It is the most in cereals are currently underutilized; thus, there is a need to common technique used to fractionate barley (8). The abra- create or identify processing fractions that are enriched in sion of rye and barley to produce high-starch pearled grains phytosterols. In this study, pearling of hulless barley and rye was investigated as a potential process to make fractions with higher also has been used to improve fuel ethanol production (9,10). levels of phytosterols. The grains were pearled with a labora- There is a need to find new uses for the pearling fines tory-scale pearler to produce pearling fines and pearled grains. and other possible low-starch by-products remaining after were extracted by accelerated solvent extraction, and separation of the high-starch pearled grain. The objective of nonpolar lipids were analyzed by normal-phase HPLC with this study was to evaluate pearling as a potential process to ELSD and UV detection. Total analyses were performed make fractions with higher levels of phytosterols. The by GC. After a 90-s pearling, the amounts of pearling fines from cereals studied were hulless barley and rye. hulless barley and rye were 14.6 and 20.1%, respectively, of the original kernel weights. During pearling, higher levels of phytosterols and other lipids were fractionated into the fines. MATERIALS AND METHODS The contents of free sterols and sterols esterified with FA in the The grains used for pearling were a new winter hulless barley fines were at least double those in the whole grains. Pearling fines of hulless barley and rye contained >2 mg/g phytosterol variety, Doyce, released in 2003 by the Virginia Polytechnic compounds, which makes them a good source of phytosterols Institute and State University and grown in Virginia. The rye and thus valuable raw materials for health-promoting foods. variety, Flesynt, was from the North Florida Research and Ed- Paper no. L9521 in Lipids 39, 783–787 (August 2004). ucation Center, Institute of Food and Agricultural Services, University of Florida (Quincy, FL). Rye grains were also hul- less. Both grains were harvested in the 2003 season, and the Phytosterols are being studied extensively because of their moisture content of the grains ranged between 11 and 14%. positive health effects. Research projects have focused on Grains were pearled with a laboratory-scale barley pearler their biological functions, safety, and chemical and physical (30 grit carborundum stone, no. 7 mesh screen; Seedburo properties, as well as on attempts to develop new phytosterol- Equipment Co., Chicago, IL). For each pearling experiment, enriched foods (1,2). As the result of a recent workshop on 50 g of grains were pearled, producing a fraction of pearling sterols and stanols with 26 leading researchers as participants fines and a mixture of broken kernels and pearled grains. (i.e., the Stresa Workshop), an extensive review was pub- The grains were first pearled sequentially to learn how phyto- lished on the effects of using phytosterols and stanols to con- sterols were localized in the kernels and to determine a relevant trol serum and the safety of phytosterol and stanol time for the production of pearling fractions. Sequential pearling enrichments in foods (3). The authors concluded that daily consisted of five steps of 30 s each. After each pearling, fines consumption of 2 g of sterols or stanols decreases serum LDL were separated from the broken kernels by passing them cholesterol levels by approximately 10%. through an 18-mesh sieve and collected as the product. Pearled Cereals are considered to be a good source of dietary grains and broken kernels were combined and pearled again. To phytosterols. Although their levels in whole grains are mod- obtain enough material, each pearling sequence was repeated erate (0.4–1.2 mg/g) (4,5), the total amount of phytosterols is five times, and the five fractions of pearling fines were extracted significant because of the large amounts of cereals consumed. and analyzed separately. In the second experiment, the grains Phytosterols, like many other bioactive compounds (e.g., to- were pearled for 90 s. Pearling fines and pearled grains from copherols, tocotrienols, and ), are unevenly distributed three separate pearlings were combined and subjected to further analysis. Each pearling experiment was performed in duplicate. *To whom correspondence should be addressed at Dept. of Applied Chem- istry and Microbiology, Latokartanonkaari 11, P.O. Box 27, FIN-00014 Uni- The grains and pearled grains were ground to 20 mesh in a versity of Helsinki, Finland. E-mail: anna-maija.lampi@helsinki.fi Wiley mill (Thomas Scientific, Swedesboro, NJ) before

Copyright © 2004 by AOCS Press 783 Lipids, Vol. 39, no. 8 (2004) 784 A.-M. LAMPI ET AL. extraction. The pearling fines were extracted without addi- the study and were 1.8 min for sterols esterified with FA (St- tional grinding. Lipids were extracted using a Dionex ASE FA), 4.5 min for TAG, 9.2–10.6 min for FFA, 21.2 min for 200 accelerated solvent extractor (ASE) (Sunnyvale, CA) free stanols, 22.0 min for free sterols, and 26.6 min for sterols with 2.0-g sample sizes and 11-mL extraction vessels as esterified with (St-Fer). All three lipid extracts for reported earlier (11). In this study, the extraction mixture each sample were analyzed for nonpolar lipids. consisted of hexane and isopropanol (3:2, vol/vol) (12). The Total phytosterols of the grains and pearling fines were an- extracts were used for gravimetric measurements of lipid ex- alyzed by GC using FID after acid and alkaline hydrolysis (5). tracts and nonpolar lipid analyses by HPLC (13). The extracts Pearling fractions were also analyzed for moisture and ash were dried under nitrogen at ≤40°C and weighed for total ex- using AACC official methods AACC 44-15A and AACC tractable lipids. The extracts were redissolved in hexane for 08-01 (14), respectively. Nonpolar lipid and total phytosterol nonpolar lipid analysis and filtered through 0.2-µm polyvinyli- results are presented as means and SD derived from three sub- dene fluoride filters (Acrodisc LC 13; Pall Gelman Labora- samples, and moisture and ash are given as means derived tory, Ann Arbor, MI) when needed. Each grain sample was from two subsamples. extracted in triplicate. In each extraction batch, whole-grain rye flour (Hodgson Mill Inc., Effingham, IL) was included as RESULTS AND DISCUSSION an in-house reference sample to monitor the extraction proce- dure. Extraction efficacy was also verified by spiking the Evaluation of the lipid analysis method. Reproducibility of same flour with (95%; Sigma Chemical Co., St. the lipid extraction method was examined by analyzing non- Louis, MO) and calculating its recovery. polar lipids of whole-grain rye flour at least once in each ex- Nonpolar lipids were analyzed by normal-phase HPLC traction batch during the study (N = 23). The contents of with LiChrosorb DIOL (5 µm, 3 × 100 mm) columns TAG, St-FA, stanols, sterols, and St-Fer were 3.65 ± 0.18, (Chrompack, Raritan, NJ) using instruments and running 0.74 ± 0.03, 0.06 ± 0.01, 0.20 ± 0.01, and 0.06 ± 0.01 mg/g, conditions as presented by Moreau et al. (13) except that the respectively. Recovery of spiked stigmasterol from the flour gradient was slightly modified. The linear gradient elution was 98% (N = 6). Detector responses of the nonpolar lipid consisted of three steps followed by a 20-min stabilization pe- analysis remained stable; the contents of TAG, St-FA, and St- riod. In step 1 (0–8 min), the eluent consisted of 100% sol- Fer of corn fiber oil were 656 ± 22 mg/g (N = 29), 44.7 ± 2.9 vent A (hexane with 0.1% ); in step 2 (8–10 min), mg/g (N = 59), and 44.9 ± 2.0 mg/g (N = 59), respectively. the proportion of solvent B (hexane with 1% isopropanol) Sequential pearling. When hulless barley and rye were was increased to 25%; and in step 3 (10–40 min), the eluent pearled sequentially, the pearling fine yields were approxi- consisted of 75% solvent A and 25% solvent B. The flow rate mately 7 and 5%, respectively, from each 30-s pearling step. was 0.5 mL/min. All lipids were detected using ELSD (All- Barley grains contained free sterols and St-FA (Fig. 1). The tech-Varex Mark III; Alltech Assoc., Deerfield, IL) except for total amount of sterol compounds in the whole barley grains phytosterols esterified with ferulic acid, which were detected was 0.70 mg/g. The highest level, 2.8 mg/g, was obtained in using UV at 280 nm. An external standard method was used the fines from the first pearling step. The total sterol content of for quantification (13). Performance of the nonpolar lipid the fines decreased with each pearling step. Even the fifth fines HPLC was checked daily using corn fiber oil (13). The reten- had 2.2 times the amount of sterol compounds as the whole tion times of nonpolar lipid classes remained stable during grains. The pearled barley grains still contained 65 and 80% of

FIG. 1. Distribution of nonpolar sterol classes in hulless barley and its products from five 30-s sequential pearling steps. Values are given as means of two replicate experiments. St-FA, sterols esterified with FA; St, free sterols and stanols.

Lipids, Vol. 39, no. 8 (2004) PEARLING OF BARLEY AND RYE 785 the free sterol and St-FA levels found in the whole grains. Non- >60% of the extractable lipids (Tables 1–3). After these polar lipid profiles showed that free sterols were more concen- lipids, the amounts of St-FA and free sterols were greatest. It trated in the outer layers of the kernels than were St-FA. should be remembered that, in addition to the nonpolar lipid Rye grains contained free sterols, free stanols, St-FA, and classes, the lipid extracts also contained some polar lipids and St-Fer (Fig. 2). The total amount of sterol compounds in the nonlipid compounds that co-extracted under ASE conditions. whole grains was 1.1 mg/g. Phytosterols were concentrated The pearling fines of hulless barley were rich in lipids. The in the pearling fines. All pearling fines contained ≥1.9 mg/g sum of TAG and FFA accounted for 5.4% of the mass of fines. sterol compounds, with the highest level being found in the The contents of phytosterol compounds were also clearly second pearling fines. After five steps, the pearled rye grains greater in the fines than in the whole grains: 1.18 and 0.43 mg/g still contained 63 and 61% of the free sterol and St-FA levels for St-FA, and 0.96 and 0.25 mg/g for free sterols, respectively. found in the whole grains. During the pearling of rye, the phytosterol compounds were The levels of sterol compounds decreased more in hulless fractionated into fines and pearled grains in a ratio similar to barley than in rye when comparing the inner layers of the ker- that of hulless barley. In the rye pearling fines, the level of St- nels to the outer ones (Figs. 1, 2). In both cereals, the sterol FA, 1.34 mg/g, was double that of the whole grains, and the levels were at least twice as concentrated in fines after the levels of free sterols increased almost threefold to 0.93 mg/g. third pearling step than the whole grains. Thus, one 90-s Although St-Fer were the least abundant sterol class in pearling was chosen for further experiments to produce whole-grain rye, 0.07 mg/g, their presence is important, be- pearling fines with high phytosterol and lipid contents at a cause they are considered to be potent antioxidants and are reasonable extraction rate. present in a number of cereals, e.g., corn, , , and rye Products from a 90-s pearling. The yields and composi- (17–20). St-Fer levels in the pearling fines were only slightly tion of pearling fines and pearled grains from two replicate higher than those in the whole and pearled grains, which does pearling experiments of each grain were similar (Table 1). not support earlier findings that the ferulates are concentrated The amounts of pearling fines of hulless barley and rye were in the outer and especially in the aleurone layer of the kernels 14.6 and 20.1% of the whole grains, and the percentages of (19,20). The small differences in St-Fer levels might be partly extractable lipids in the products were 7.08 and 5.08%, re- due to their overall low level in the extracts and the analytical spectively. Extractable lipid contents of the products were uncertainty derived from that. In the sequential pearling ex- comparable with those in earlier studies (15,16). Since the periments, however, St-Fer in the fines were clearly greater yield of pearling fines from hulless barley was lower than that than in the pearled grains. St-Fer were not found in any barley from rye, the pearling fines of barley contained proportion- products, which is in accordance with earlier studies (19). ally more lipid-rich outer layers of the kernels than those of After a 90-s pearling step, the pearled barley and rye rye, and the lipid content of the pearling fines of barley was grains still contained phytosterol compounds at levels of 0.5 higher. Similarly, the ash contents showed that the pearling and 0.8 mg/g, respectively, which means that even the fines of barley were enriched in ash in the outer layers, be- pearled grains have an important impact on our natural di- cause the ash content was 2.2 times higher than that of the etary phytosterol intake. If phytosterols were isolated for the , whereas in rye the ratio was 1.6. enrichment of their levels in regular foods, the pearling fines Whole grains of hulless barley and rye contained 1.1% from hulless barley or rye would be a better source of phyto- TAG and 0.1% FFA, two major lipid classes, and contributed sterols than the whole grains.

FIG. 2. Distribution of nonpolar sterol classes in rye and its products from five 30-s sequential pearling steps. Values are given as means of two replicate experiments. St-Fer, sterols esterified with ferulic acid; for other abbreviations, see Figure 1.

Lipids, Vol. 39, no. 8 (2004) 786 A.-M. LAMPI ET AL.

TABLE 1 grains (35% in barley and 5% in rye) could thus occur as glyco- Characterization of Two Replicate 90-s Pearlings (A and B) of Hulless sides and acylated glycosides. Steryl glycosides may contribute a Barley and Rye to ca. 20% of total phytosterols in whole-grain wheat flour (21). Sample Yield (%) Extractable lipids (%) Ash (%) Moisture (%) Three phytosterols, namely, sitosterol, , and Whole-grain stigmasterol, were the major sterols in barley and rye (Table 4), barley 100 1.95 ± 0.07 1.8 12.4 which is in accordance with earlier studies (4,5). Up to 18% of Pearling fines, A 14.5 7.04 ± 0.10 4.0 10.7 total sterols in rye consisted of saturated phytosterols, sitostanol, Pearling fines, B 14.6 7.12 ± 0.06 3.9 10.7 Pearled grains, A 85.5 1.46 ± 0.06 1.4 12.2 and campestanol. The amount of stanols in barley was lower Pearled grains, B 85.7 1.44 ± 0.02 1.4 12.3 than that in rye, which was also shown by the nonpolar lipid Whole-grain rye 100 1.84 ± 0.04 2.1 10.6 analysis. Phytosterol profiles of the pearling fines were compa- Pearling fines, A 19.8 5.11 ± 0.05 3.4 9.4 rable with those of the whole grains. In barley, the amounts of Pearling fines, B 20.5 5.05 ± 0.01 3.4 9.4 stanols in the fines were greater than those in the grains. Pearled grains, A 77.4 1.14 ± 0.02 1.7 10.7 Pearled grains, B 76.7 1.15 ± 0.02 1.7 10.3 Potential food uses of pearling fines. Pearling fines are usually considered by-products of the milling and/or pearling aData are mean ± SD (N = 3) or mean (N = 2). industry, with the high-starch fractions as the main products. The phytosterol contents obtained from the nonpolar lipid However, the by-products contain higher levels of many valu- analyses were consistent with the total phytosterol analysis re- able bioactive compounds (e.g., and , and sults obtained by GC. When sterol contents present in free dietary fiber) than the refined products. This study showed that sterols, St-FA, and St-Fer were calculated, most of the total the pearling fines of hulless barley and rye contained >2 mg/g phytosterols in barley and rye grains could be attributed to phytosterol compounds, which makes them a reliable and in- these classes. A small proportion of total phytosterols in the expensive source of phytosterols. Recent studies have shown

TABLE 2 Nonpolar Lipids of Hulless Barley and Its Products After 90-s Pearlinga Lipid contents (mg/g)

Sample TAG FFA St-FA Stanols Sterols St-Fer Whole grains 11.4 ± 0.2 1.00 ± 0.03 0.43 ± 0.01 +b 0.25 ± 0.00 NDc Pearling fines, A 50.4 ± 1.2 3.08 ± 0.04 1.16 ± 0.02 + 0.94 ± 0.01 ND Pearling fines, B 50.8 ± 1.0 3.17 ± 0.08 1.19 ± 0.01 + 0.99 ± 0.03 ND Pearled grains, A 7.35 ± 0.5 0.81 ± 0.02 0.34 ± 0.01 + 0.20 ± 0.01 ND Pearled grains, B 7.1 ± 0.2 0.80 ± 0.01 0.33 ± 0.00 + 0.20 ± 0.00 ND aA and B refer to replicate pearling experiments. Data are mean ± SD (N = 3). b+, present at <0.05 mg/g. cND, not detected. TABLE 3 Nonpolar Lipids of Rye and Its Products After 90-s Pearlinga Lipid contents, (mg/g)

Sample TAG FFA St-FA Stanols Sterols St-Fer Whole grains 11.1 ± 0.2 1.09 ± 0.02 0.62 ± 0.02 0.09 ± 0.00 0.33 ± 0.00 0.07 ± 0.01 Pearling fines, A 34.0 ± 0.7 1.87 ± 0.04 1.33 ± 0.06 +b 0.92 ± 0.01 0.07 ± 0.01 Pearling fines, B 34.4 ± 0.3 1.94 ± 0.05 1.34 ± 0.01 + 0.94 ± 0.01 0.08 ± 0.02 Pearled grains, A 4.75 ± 0.1 1.04 ± 0.02 0.45 ± 0.02 0.06 ± 0.00 0.20 ± 0.00 0.07 ± 0.00 Pearled grains, B 4.7 ± 0.2 1.06 ± 0.03 0.45 ± 0.00 0.06 ± 0.00 0.20 ± 0.00 0.06 ± 0.00 aA and B refer to replicate pearling experiments. Data are mean ± SD (N = 3). b+, present at <0.05 mg/g.

TABLE 4 Total Phytosterol Compositions of Whole Grains and 90-s Pearling Fines of Hulless Barley and Ryea Total sterol contents, (µg/g)

Sample Campesterol Campestanol Sitosterol Sitostanol Stigmasterol Minor sterolsb Total phytosterols Whole-grain barley 181 ± 2 11 ± 0 476 ± 1 5 ± 0 39 ± 0 86 ± 1 797 ± 4 Pearling fines, A 391 ± 8 40 ± 0 925 ± 20 26 ± 0 120 ± 1 221 ± 9 1732 ± 38 Pearling fines, B 397 ± 5 40 ± 0 934 ± 10 25 ± 0 121 ± 1 227 ± 1 1744 ± 14 Whole-grain rye 133 ± 1 68 ± 1 452 ± 4 94 ± 1 29 ± 1 111 ± 1 886 ± 7 Pearled fines, A 297 ± 3 100 ± 2 795 ± 9 122 ± 2 74 ± 1 199 ± 3 1587 ± 19 Pearled fines, B 300 ± 6 105 ± 3 804 ± 12 126 ± 2 73 ± 1 212 ± 11 1620 ± 33 aA and B refer to replicate pearling experiments. Data are mean ± SD (N = 3). bMinor sterols include, e.g., ∆5- and ∆7-avenasterols, ∆7-stigmastenol, and stigmastadienol.

Lipids, Vol. 39, no. 8 (2004) PEARLING OF BARLEY AND RYE 787 that the phytosterol levels present in natural foods might also Schaafsma, G., eds.), pp. 135–140, Wageningen Academic Pub- contribute to lowering cholesterol absorption (22,23). It also lishers, Wageningen. has been suggested that when phytosterols are combined with 8. Izydorczyk, M.S., Symons, S.J., and Dexter, J.E. (2002) Frac- tionation of Wheat and Barley, in Whole-Grain Foods in Health other bioactive compounds, they may have a greater impact and Disease (Marquart, L., Slavin, L., and Fulcher, R.G., eds.), on health. Moreover the LDL-lowering effect of phytosterols pp. 47–82, American Association of Cereal Chemists, St. Paul, MN. might be enhanced by other cholesterol-lowering compounds 9. Sosulski, K., Wang, S., Ingledew, W.M., Sosulski, F.W., and such as soluble , , and soy protein (24), which Tang, J. (1997) Preprocessed Barley, Rye, and Triticale as a may make the pearling fines valuable raw materials for health- Feedstock for an Integrated Fuel Ethanol-Feedlot , Appl. Biochem. Biotechnol. 63–65, 59–70. promoting foods. More research is needed to understand the 10. Wang, S., Sosulski, K., Sosulski, F., and Ingledew, M. (1997) interactions between phytosterols and dietary fibers, and to de- Effect of Sequential Abrasion on Starch Composition of Five velop processes to enable the efficient utilization of the milling Cereals for Ethanol , Food Res. Intl. 30, 603–609. industry by-products to yield improved food applications. 11. Moreau, R.A., Powell, M.J., and Singh, V. (2003) Pressurized In addition to possible food uses of barley pearling fines, a Liquid Extraction of Polar and Nonpolar Lipids in Corn and with Hexane, Methylene Chloride, Isopropanol, and Etha- new type of edible oil also could be obtained by the extraction nol, J. Am. Oil Chem. Soc. 80, 1063–1067. of barley pearling fines. (Pearling fines contain about 4.5 times 12. Hara, A., and Radin, N.S. (1978) Lipid Extraction of Tissues as much oil as whole grains.) From the data on pearling fines, with a Low-Toxicity Solvent, Anal. Biochem. 90, 420–426. we estimate that a “barley pearling fine oil” contains about 3% 13. Moreau, R.A., Powell, M.J., and Hicks, K.B. (1996) Extraction total phytosterols, which is several-fold higher than the levels and Quantitative Analysis of Oil from Commercial Corn Fiber, J. Agric. Food Chem. 44:2149–2154. of phytosterols in typical commercial oils. Since 14. American Association of Cereal Chemists (2000) Approved Ostlund et al. (25) demonstrated that the endogenous phyto- Methods of the AACC, 10th edn., AACC, St. Paul, MN. sterols in refined had a significant cholesterol-lowering 15. Nilsson, M., Åman, P., Härkönen, H., Hallmans, G., Bach effect, it is reasonable to assume that an oil from barley fines Knudsen, K.E., Mazur, W., and Adlercreutz, H. (1997) Content may have similar health-promoting properties. of and in Roller Milled Fractions of Rye, J. Sci. Food Agric. 73, 143–148. 16. Yeung, J., and Vasanthan, T. (2001) Pearling of Hull-less Bar- ACKNOWLEDGMENTS ley: Product Composition and Gel Color of Pearled Barley Flours as Affected by the Degree of Pearling, J. Agric. Food Collaboration between the Department of Applied Chemistry and Chem. 49, 331–335. Microbiology (University of Helsinki) and the USDA/ARS/ERRC 17. Seitz, L.M. (1989) Stanol and Sterol Esters of Ferulic and was financially supported by the National Technology Agency of p-Coumaric Acids in Wheat, Corn, Rye, and Triticale, J. Agric. Finland. The authors would like to thank Drs. Mark Vaughn, Carl Food Chem. 37, 662–667. Griffey, and Wynse Brooks for providing us with the barley grains 18. 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