Plan I Foods lor Human NUlrilion 45: 53-61, 1994. ~: 1994 Kluirer Academic Publishers. Prillled ill Ihe Nelherlands. 7125

Oat p-glucan-amylodextrins: Preliminary preparations and biological properties

G.E. INGLETT1* & R.K. NEWMAN2 I Biopolymer Research Unil. iVaiional eenler for Agriculrural Ulilizalion Research, USDA­ ARS, 1815 iV. Universily SI.. Peoria.IL 61604 USA; lDeparlmellI oIPlanl and Soil Science. Monuma Slate University. Bozeman, MT 59717 USA ("'author for correspondence)

Received II February 1992: accepted in revised form IS June 1992

Key words: Amylodextrin, fJ-glucan. , Cholesterol, replacement

Abstract. Amylodextrins with soluble fJ-glucan contents from I to 10% were prepared from oats and the hypocholesterolemic properties of the latter were evaluated. The products are called OATRIM and can lower blood cholesterol by replacing animal rich in cholesterol in food products and, possibly, by the action of fJ-glucan in the body after consumption. In the chick model. decreased total blood cholesterol also resulted in increases of HDL choles­ terol and decreases of LDL cholesterol. Processing conditions \vere found that gave the maximum amount of fJ-glucan and desirable fat-replacement qualities with the least amount of color and flavor.

Introduction

Elevated blood cholesterol has been found associated with risk of coronary heart disease (Berenson et aI., 1978; American Heart Association, 1980). The National Research Council recommends limiting fat consumption to 30% or less of total calories and saturated fat consumption to 10% of calories to reduce the risk of atherosclerosis, cancer and other chronic diseases. In addition to dietary fat-reduction, caloric and cholesterol-lower­ ing are considered important factors in health. is considered to be the soluble and insoluble components of food that are not digested by in the human gastrointestinal tract. The primary sources of dietary fiber include such cell wall materials as cellulose, hemicelluloses, lignin, and pectins, along with gums and mucilages. Dietary fiber has been considered as an important food com­ ponent since early times. Burkitt et al (1972) and Trowell (1972) concluded

The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned. 54 that dietary fiber has a role in the prevention of certain large-intestine diseases, including cancer of the colon and diverticulitis. Burkitt also men­ tioned that the serum cholesterol rises when dietary fiber is removed from the diet, and that eating a fiber-rich diet lowers serum cholesterol. It is known that all dietary fiber is not the same and that different fibers provide different health benefits. For example, wheat is very rich in insoluble dietary fiber (mainly cellulose and hemicelluloses) and is excellent for decreasing the transit time offood through the digestive tract (Anderson et aL 1979) and increasing food bulk. The soluble dietary fibers are reported to reduce total plasma cholesterol (Munoz et aI" 1979). Over the years, numerous experiments with animals have shown that fiber has a strong hypocholesterolemic effect (DeGroot et aI., 1963; Fisher et aI., 1967). Ander­ son et al. (1984) have confirmed hypocholesterolemic effects of oats in humans. , or rolled oats, and especially oat bran are excellent sources of this soluble fiber which is effective in lowering cholesterol levels. Moreover, oat fiber reduces the amount of low density lipoprotein (LDL) without lowering the beneficial high density lipoprotein (HDL). In fact, oat bran fed to humans reduced serum cholesterol concentration by 19% and calculated LDL cholesterol by 23 % (Anderson et aI., 1984). Other water-soluble fibers; e.g., pectin and guar gum, can lower serum cholesterol, but their use is frequently accompanied by undesirable side effects such as nausea and vomiting (Anderson et al., 1984). Amylodextrins are produced from corn starch by acid or hydrolysis to give a variety of products, with physical properties dependent on the degree of conversion. Acid conversions are known to give uniform distribution of hydrolysate fragments because of the random cleavages of the starch molecule, whereas enzymic treatments result in variations in amounts of the different oligomer fragments (Inglett 1987, 1990). Various amylolytic enzymes are used in the thinning of liquefaction of corn starch and in the production of low conversion starch hydrolysates which are known in the trade as maltodextrins or corn syrup solids, depending upon the degree ofhydrolysis (Morehouse et al., 1972). Other sources ofstarch for commercial products are tapioca, potato and rice. Whole have also been subjected to starch-hydrolyzing conditions and have yielded, for example, a whole-grain hydrolyzed product (Conrad, 1983) and a ready-to­ eat, enzyme-saccharified cereal (Fulger and Gum, 1987). 55

Materials and methods

Proximate analyses for moisture, , and ash were obtained by official procedures of the AACC (1983) and total lipid by AOCS (1980). The automated high performance liquid chromatographic (HPLC) system used to analyze the hydrolysates has been reported earlier (Inglett, 1987). Com­ mercially available amylases were used in these studies: B. stearother­ mophilus CI.-amylase, 'G-zyme G995' (Enzyme Bio-Systems Ltd., Interna­ tional Plaza, Englewood, NJ); 'Enzeco Thermolase' (Enzyme Development Corp., New York, NY); and B. lichen(formis CI.-amylase, 'Taka Therm L-340' (Solvay Enzymes, Elkhart, IN).

Laboratory procedure

For each preparation, 100 g (dry basis, db) of oat (The Quaker Oats Company, Cedar Rapids, IA) was slurried in 400 ml of water containing

25 ppm of calcium (0.09 giL CaCl2 ·2H2 0) and gelatinized by passage through a steam injection cooker at 138-143 DC (30-40 psi of steam pres­ sure). The gelatinized mixture was adjusted to the desired pH with 1.0 N sodium hydroxide. 'G-zyme G995' was added to the mixture at 95 DC in an amount sufficient to provide 1 unit per g ofoat flour, where 1 unit ofamylase activity is the amount of enzyme required to hydrolyze 10 mg of starch per minute under specified conditions. After 5 min ofstirring at 95 DC, the starch was liquified, and the enzyme was inactivated by passing the mixture through a steam injection cooker. The mixture was then allowed to cool to about 70 DC, when it was centrifuged 30 min at 5000 rpm. The water-soluble fiber product in the supernatant solution was recovered by decanting the solution and freeze-drying. The insoluble residue obtained from centrifuging was removed and air dried. The degree of polymerization (DP) distribution of starch oligomers in the products was determined by high-pressure liquid chromatography (Inglett, 1987).

Large scale treatment of oat bran

Four kg of oat bran (National Oats Company Cedar Rapids, IA) in a large Sigma mixer was slurried in 28 L ofwater containing 50 ppm ofcalcium. The slurry was adjusted to pH 6.0 with 1.0 N sodium hydrozide, and 'Taka Therm L-340' was added in an amount sufficient to provide 1632 Modified Wohlgemuth Units (MWU) per g ofsubstrate, where I MWU is that activity which will dextrinize 1mg ofsoluble starch to a defined size dextrin in 30 min under specified conditions as determined by Solvay Enzymes, Elkhart, IN. 56

During a period ofabout 10-15 min, the temperature was increased to about 95 DC with steam heat. After 20 min, the enzyme was inactivated by passing the mixture through a steam injection cooker. The warm slurry was centrifuged at 15,000rpm in a large 'Sharples' centrifuge to separate the soluble from the insoluble components. The percent oligomer composition of the solubles was: DP > 9,32.3; DP 9, 0; DP 8, 0; DP 7,1.0; DP 6,13.0; DP 5, 13.9; DP 4, 6.4; DP 3, 13.0; DP 2, 12.1; and DP 1, 5.6.

Large scale treatment ofoat flour

Three kg ofoat flour (National Oats Company) was slurried in 18 L ofwater containing 50ppm of calcium. The pH of the slurry was 5.75. After gelatinization by passage of the mixture through a steam injection cooker, the slurry was collected in a 30-gal steam-heated kettle. 'Enzeco Thermolase' was added to the slurry in an amount sufficient to provide 1 unit per g ofoat flour. After 5 min of stirring at 80-90 DC, the enzyme was inactivated by passing the slurry through a steam injection cooker. The warm slurry was centrifuged at 15,000rpm in a large 'Sharples' centrifuge to separate the soluble and insoluble components. The products were dried separately on hot rolls. Analysis of the soluble component indicated an oligomer com­ position of 98% DP 9 and larger.

Results and discussion

Water-soluble dietary fiber compositions were recovered from such milled products as oat bran and oat flour after enzymatic hydrolysis of these substrates with cr.-amylases (Inglett, 1990, 1991a, 1991b). The resulting soluble dietary fiber compositions that separated with the soluble hydrolyzate fractions were colorless and devoid of undesirable flavors. The soluble fiber compositions could, therefore, be suitable for use in a wide variety of foods (Inglett and Warner, 1990). The slurried substrate was gelatinized prior to enzymatic treatment. The ungelatinized slurry or the gelatinized dispersion was adjusted to about pH 5.5-7.5, preferably about 6.0, with sodium hydroxide or other alkali, and the cr.-amylase was added. Among the wide selection of cr.-amylases that could be used, the commer­ cial cr.-amylases referred to as 1,4-cr.-D-glucan glucanohydrolases which have the essential enzymatic characteristics of those produced by the Bacillus stearothermophilus strains were found to be satisfactory. Other commercial sources of this enzyme included organisms such as B. subtilis and its genetic- 57 ally modified form to express the thermostable a-amylase of B. stearo­ thermophilus. Other suitable a-amylases were those having the essential enzymatic characteristics of those produced by B. licheniformis. Of course, any a-amylase which is useful in the thinning of the starch could be employed. The conditions of enzyme treatment, including the enzyme concentration and the time and temperature of reaction, were selected to achieve liquefac­ tion of the starch in the substrate to the extent that the soluble fiber bound by the cellular matrix was substantially completely liberated into solution. When thermostable a-amylase was used, a preferred treatment temperature was in the range of 70-100 DC. At these temperatures, gelatinization of the starch in the substrate occurred concurrently with the hydrolysis. Other a-amylases could be used at lower temperatures. The duration of the treat­ ment at the desired conversion temperature depended in the desired product properties and generally can range from about 1 to 60 min. After completion of the enzymatic hydrolysis, the enzyme was inac­ tivated; e.g., by passing the mixture through a steam injection cooker at a temperature of about 140 DC. Alternatively, the enzyme could be inactivated by acidification (pH 3.5-4.0) at 95 DC for about 10 min. Optional neutraliza­ tion with alkali increases the salt concentration of the product. After inac­ tivation, the soluble fraction, comprised of the soluble oat fiber and amylodextrin, was separated from the insoluble residue by centrifugation. Water was then removed from the soluble fraction in the centrifugate by spray-drying, drum-drying, or freeze-drying to recover the soluble oat fiber product. Water was removed from the fractions by conventional techniques after the insoluble residue was separated. The water-soluble material contains amylodextrins, f3-g1ucan, some lipid, protein, and minerals. Amylodextrins containing the soluble-fibers were white, non-gritty preparations that are devoid of undesirable flavors. Since the soluble-fibers of oats are principally f3-g1ucan, these compositions are referred to as oat f3-glucan-amylodextrins. For purposes of brevity and to focus on the nutritional aspects of these products, these products are called OATRIM. OATRIM-l was prepared from debranned whole oat flour, OATRIM-5 was prepared from whole oat flour. Oat bran was used to prepare OATRIM-IO. The number after OATRIM refers to the percentage of f3-g1ucan present in the preparation on a dry basis. The compositions of the various OATRIM products are shown in Table 1. Characterizations (Table 2) of insoluble fiber products from the conver­ sion mixtures revealed high proportions ofinsoluble fiber, protein and lipid. The protein was believed to complex with the lipid and to become 58

Table I. OATRIM compositions'"

Component OATRIM-l OATRIM-5 OATRIM-1O %. db %. db %. db

Fat (Crude) 0.3 1.4 0.6 Protein (Nitrogen x 6.25) 1.9 5.0 4.0 Minerals (Ash) 1.4 3.1 4.2 f3-g1ucan 1.6 5.8 10.2 Amylodextrins (by difference) 94.8 84.7 81.0

"'See text for details of preparation of soluble fiber products. OATRIMS-I. -5 and -10 were prepared by respective treatment of debranned whole oat flour. whole oat flour and oat bran.

Table 2. Insoluble fiber products (IFP)

Residues from preparation of:

Component OATRIM-I OATRIM-5 OATRIM-IO IFP. %. db IFP. %. db IFP. %. db

Fat (Crude) 18.5 14.0 8.1 Protein (Nitrogen x 6.25) 36.3 42.3 46.0 .., , Minerals (Ash) ~ ..) 1.9 4.8 Insoluble Fiber (by difference) 42.9 41.8 41.1 insolubilized by heat. The residue also contained the insoluble fiber and the majority of the flavor and color components. When Bacillus stearothermophilus a-amylase was added in varying amounts to oat flour, as shown in Table 3, the DP distribution of starch oligomers in the products, as determined by high-pressure liquid chromatography (Inglett, 1987), was found to vary. The data in Table 3 show that lower amounts of enzyme amounts result in higher DP starch oligomers. Since OATRIM was obtained as a white, smooth-textured product devoid of undesirable flavor, it is suitable for use as a functional and nutritional component of many foods including dairy, meat, and bakery products where consumer preference dictates the virtual absence of the flavor, color, and grittiness inherent to the oat flour or bran.

Hypocholesterolemic properties of OATRJjI;j

OATRIM-I0, containing 10% fJ-glucan, was tested in the chick model at Montana State University (Newman, personal communication, 1990). Hub­ bard broiler cockerel chicks were received the day after hatching and fed a 23% protein corn/soy isolate diet for adjustment and growth for two days, followed by a 7-day period with 0.5% cholesterol added to the diet, to create a hypercholesterolemic condition. Chicks were weighed, wing-banded to 59

Table 3. Influence of amylase activity* on oat flour amylodextrin composition

Enzyme concentration DP> 9 (units/g substrate) (% of total oligomers)

6 93.0 12 47.7 24 36.5

*Conditions given in text. assign individual identification, and assigned to two groups of 8 each. One group continued on the corn/soy isolate diet as a control, and the other group was fed this diet with 26.4% OATRIM-lO substituted for an equal quantity ofcorn. Soluble f3-glucan content ofthe chick diet was 2.2%, based on an 8.4% concentration in the OATRIM. At the end of 10 days, chicks were fasted for 12 h and final live weights recorded. Blood samples were taken from brachial veins and plasma lipid profile analyses completed by use of a Kodak Ektachem DT 60 analyzer (Allain et aL 1974; Megraw et aI., 1979).

Table 4. Body weight and serum lipids of chicks fed OATRIM for 10 days

Control OATRIM Probability

Body weight gain. g 301.1 201.2 0.0006 Total plasma cholesterol. mg/dl 185.0 151.6 0.0153 HDL-cholesterol. mg/dl 78.6 92.5 0.0743 LDL-cholesterol, mg/dl 95.4 49.9 0.0002 Triglycerides. mg/dl 55.0 46.5 0.0874

The results are shown in Table 4. Chicks fed OATRIM gained significant­ ly less (p < 0.0006) than controls, which could have influenced the plasma lipids in part. There were significant reductions in total cholesterol (p < 0.0153) and LDL-cholesterol (p < 0.0002) in chicks fed OATRIM compared to controls. HDL-cholesterol levels were numerically higher in OATRIM-fed chicks, but the difference was not significant. There was no significant difference in triglycerides between the two groups. Although the number of experimental animals was small, the results show promise of the healthful benefits of OATRIM. Further studies are indicated.

Summary

Hypocholesterolemic f3-glucan-amylodextrins were prepared by the treat­ ment of oat flour and bran products with C(-amylases under specially controlled conditions. Under these conditions, oat starch was converted into 60

amylodextrins, which go into solution along with the soluble beta-glucans. These white-colored, nearly tasteless solids did not have noticeable cereal flavor, and lack the bitter and gritty constituents normally present in oat flours and bran. As shown in chick-feeding experiments, the j3-glucan com­ ponent of these new ingredients could contribute hypocholesterolemic effects to foods. The oat j3-glucan-amylodextrins, called OATRIM, are new ingredients used as fat substitutes in many food applications.

Acknowledgement

My personal thanks for the technical assistance of Greg Friestad, Harold Griffin, Mike Jakoby, Gary Kuzniar, Sean Mellican, Ron Montgomery, Paulette Smith, Jim Van Cauwenberge, and Kathy Warner at the National Center for Agricultural Utilization Research.

References

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