Influence of lignification and feruloylation of maize cell walls on the adsorption of heterocyclic aromatic amines

Both epidemiological and experimental data indicate that a diet rich in fiber may reduce cancer risk. One possible mechanism is by adsorbing carcinogens and transporting them out of the body without metabolic activation. We investigated the role of fiber lignification and feruloylation on the adsorption of four of the most relevant heterocyclic aromatic amines in food: 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and 2-amino-9H-pyrido[2,3-b]indole (AalphaC). Adsorption experiments, under conditions mimicking the small intestine, were carried out using nonlignified and artificially lignified primary maize walls with defined lignin and ferulate/diferulate concentrations and defined lignin compositions. Lignin concentration and composition both influenced the adsorption of heterocyclic aromatic amines, especially the more hydrophobic types. Heterocyclic aromatic amine adsorption increased with lignin concentration. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-9H-pyrido[2,3-b]indole were better adsorbed by guaiacyl-rich lignins, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by syringyl-rich lignins, whereas the adsorption of 2-amino-3-methylimidazo[4,5-f]quinoline was not clearly influenced by lignin composition. Nonlignified cell walls adsorbed lesser amounts of heterocyclic aromatic amines. Variations in cell wall feruloylation had no effect on heterocyclic aromatic amine adsorption.     

Physicochemical and structural characterization of alkali soluble lignins from oil palm trunk and empty fruit-bunch fibers.

Six alkali soluble lignin fractions were extracted from the cell wall materials of oil palm trunk and empty fruit-bunch (EFB) fibers with 5% NaOH, 10% NaOH, and 24% KOH/2% H(3)BO(3). All of the lignin fractions contained rather low amounts of associated neutral sugars (0.8-1.2%) and uronic acids (1.1-2.0%). The lignin fractions isolated with 5% NaOH from the lignified palm trunk and EFB fibers gave a relatively higher degree of polymerization as shown by weight-average molecular weights ranging between 2620 and 2840, whereas the lignin fractions isolated with 10% NaOH and 24% KOH/2% H(3)BO(3) from the partially delignified palm trunk and EFB fibers showed a relatively lower degree of polymerization, as shown by weight-average molecular weights ranging between 1750 and 1980. The results obtained by alkaline nitrobenzene oxidation showed that all of the lignin preparations contained a high proportion of noncondensed syringyl units with small amounts of noncondensed guaiacyl and fewer p-hydroxyphenyl units. The lignin fraction extracted with 5% NaOH from the lignified EFB fiber was mainly composed of beta-O-4 ether-linked units. Small amounts of 5-5', beta-5, and beta-beta' carbon-carbon linkages were also found to be present between the lignin structural units. Further studies showed that uronic, p-hydroxybenzoic, and ferulic acids in the cell walls of palm fibers were esterified to lignin.     

Methyl esterification divergently affects the degradability of pectic uronosyls in nonlignified and lignified maize cell walls

Nonlignified cell walls from Zea mays (L.) cell suspensions were incubated with and without pectin methylesterase (PME) and a portion were artificially lignified to assess how methyl esters influence the release of pectic uronosyls and total sugars from cell walls by fungal enzymes. Treatment with PME reduced uronosyl concentrations from 97 to 92 mg/g, reduced uronosyl methylation from 57% to 21%, and increased Klason lignin concentrations in artificially lignified cell walls from 99 to 116 mg/g. Although PME treatment slightly enhanced uronosyl release from nonlignified cell walls, it reduced uronosyl release from artificially lignified cell walls by 55% after 4 h and by 7% after 72 h of enzymatic hydrolysis. Pectin hydrolysis in PME treated cell walls was probably impaired by enhanced benzyl ester cross-linking of uronosyls to lignin via quinone methide intermediates. Variations in uronosyl methylation had little effect on the overall release of total sugars from cell walls.     

Chemical composition and enzymatic degradability of xylem and nonxylem walls isolated from alfalfa internodes

During plant maturation, degradability of alfalfa (Medicago sativa L.) stems declines due to accumulation of highly lignified xylary tissue. Xylem and nonxylem tissues dissected from lower alfalfa internodes were analyzed for cell wall constituents and degradability. Cell walls comprised 740 mg g(-1) of xylem and 533 mg g(-1) of nonxylem tissues. Xylem tissues contributed about 60% of the cell wall mass in internodes. Xylem walls contained 28% lignin, 4% pectin, 29% hemicellulose, and 39% cellulose as compared to 15% lignin, 25% pectin, 30% hemicellulose, and 30% cellulose in nonxylem walls. Fungal enzymes hydrolyzed 22 and 73% of the structural carbohydrates in xylem and nonxylem walls, respectively. In both cell wall fractions, the release of xylose was 56-90% lower than that of other sugars, indicating that lignin preferentially restricted xylan degradation in secondary walls and xyloglucan degradation in primary walls. Elucidation of lignin-xylose interactions may reveal strategies for improving fiber degradability of alfalfa.     

Identifying new lignin bioengineering targets: impact of epicatechin, quercetin glycoside, and gallate derivatives on the lignification and fermentation of maize cell walls

Apoplastic targeting of secondary metabolites compatible with monolignol polymerization may provide new avenues for designing lignins that are less inhibitory toward fiber fermentation. To identify suitable monolignol substitutes, primary maize cell walls were artificially lignified with normal monolignols plus various epicatechin, quercetin glycoside, and gallate derivatives added as 0 or 45% by weight of the precursor mixture. The flavonoids and gallates had variable effects on peroxidase activity, but all dropped lignification pH. Epigallocatechin gallate, epicatechin gallate, epicatechin vanillate, epigallocatechin, galloylhyperin, and pentagalloylglucose formed wall-bound lignin at moderate to high concentrations, and their incorporation increased 48 h in vitro ruminal fiber fermentability by 20-33% relative to lignified controls. By contrast, ethyl gallate and corilagin severely depressed lignification and increased 48 h fermentability by about 50%. The results suggest several flavonoid and gallate derivatives are promising lignin bioengineering targets for improving the inherent fermentability of nonpretreated cell walls.     

In Vitro Bile Acid Binding Capacity of Milled Wheat Bran and Milled Extruded Wheat Bran at Five Specific Mechanical Energy Levels

The in vitro binding of bile acids of milled wheat bran (MWB) and milled extruded wheat bran (MEB) at five specific mechanical energy (SME) levels of 120 (MEB‐120), 177 (MEB‐177), 234 (MEB‐234), 291 (MEB‐291), and 358 (MEB‐358) Whr/kg on a fat‐free dry weight basis was determined using a mixture of bile acids secreted in human bile at duodenal physiological pH 6.3. Relative to cholestyramine (bile acid binding, cholesterol lowering drug) in vitro bile acid binding capacity on dry matter, total dietary fiber (TDF), and insoluble dietary fiber (IDF) basis was for MWB: 21, 43, 45%; the range for MEB was 18–21%, 34–41%, and 36–43%, respectively. MWB resulted in significantly higher bile acid binding than that of MEB at 120, 234, and 291 Whr/kg on a dry matter, TDF, and IDF basis. These results demonstrate the relative health‐promoting potential of MWB = MEB‐177 = MEB‐358 > MEB‐120 = MEB‐234 = MEB‐291 as indicated by the bile acid binding on a dry matter basis. Data suggest that significant improvement in health‐promoting (cholesterol‐lowering and cancer‐preventing) potential could be obtained in WB by milling (low‐cost processing) the bran to finer particle sizes and extruding (high‐cost technology). Milling WB to small particle size (weighted mean 0.508 mm) increased surface area, in addition it may have induced changes in the physical and chemical characteristics of WB or created new linkages, binding sites of the proteins, starches, and nonstarch polysaccharides, which significantly increased the bile acid binding ability of the MWB.     

In Vitro Bile Acid Binding Capacity of Wheat Bran with Different Particle Sizes

The bile acid binding capacity of wheat bran with different particle sizes was determined. Unmilled wheat bran with an average particle size of 900 μm (WB‐900), milled wheat bran at two particle sizes, 500 and 200 μm (WB‐500 and WB‐200), and all three bran samples washed with water (WWB‐900, WWB‐500, and WWB‐200) were mixed with bile acids at pH 6.3 to determine their in vitro adsorption capacity. On a dry matter basis, the order of relative bile acid binding values was WB‐900 ∼ WB‐500 > WWB‐900 > WB‐200 > WWB‐500 > WWB‐200. Data suggests that the surface area as measured by the Brunauer–Emmett–Teller (BET) method and water holding capacity may significantly affect the bile acid binding capacity of wheat bran. As the BET surface area increased with decreasing particle size, the water holding capacity and bile acid binding decreased. Bile acid binding capacity of wheat bran appears to be linked to the ability of the samples to physically adsorb the bile acids. Bile acid binding capacity significantly decreased with reduction in particle size of wheat bran after water washing.     

Tissue-specific and developmental modifications of grape cell walls influence the adsorption of proanthocyanidins

Cell wall material from Vitis vinifera L. cv. Cabernet Sauvignon grape skin and flesh was isolated at different stages of grape maturity to determine whether developmental changes in cell wall composition in different tissue types influence the binding of proanthocyanidins (PAs). Trends in cell wall adsorption of, and selectivity for, PAs were determined using two skin PAs that differed in their average molecular masses. Flesh cell walls consistently bound a higher amount of PA than those from skin. Key structural differences that reduced PA adsorption in skin cell walls by comparison with flesh cell walls were endogenously higher concentrations of insoluble PA, Klason lignin, and lower cell wall-bound protein. These differences may confer reduced flexibility and porosity of skin cell walls relative to flesh cell walls. Analysis of skin and flesh cell wall properties revealed that the onset of ripening was associated with a loss of type I arabinogalactan and galacturonic acid, which indicated a degradation of pectin within the cell wall. Flesh cell walls consistently bound PAs of larger molecular mass, and changes in PA adsorption properties after the onset of ripening were minor. For skin cell walls, adsorption of PA was lowest immediately following solubilization of galacturonic acid, and high molecular mass PAs were poorly bound. As ripening progressed, PAs of higher molecular mass were selectively adsorbed by skin cell walls, which indicates that ongoing cell wall remodeling during ripening may confer an increased porosity within the skin cell wall matrix, resulting in a greater adsorption of PA within a permeable structure.     

In Vitro Bile‐Acid‐Binding of Whole vs. Pearled Wheat Grain

Health benefits of consuming whole grains are reduced risk of heart disease, stroke, and cancer. The U.S. Health and Human Services and USDA dietary guidelines recommend consumption of 6–10 oz of grain products daily and one‐half of that amount should contain whole grains. Whole grains contain vitamins, minerals, fiber, and phytonutrients. Bile‐acid‐binding capacity has been related to cholesterol lowering potential of food fractions. Lowered recirculating bile acids results in utilization of cholesterol to synthesize bile acid and reduced fat absorption. Secondary bile acids have been associated with increased risk of cancer. Bile‐acid‐binding potential has been related to lowering the risk of heart disease and that of cancer. It has been reported that bile‐acid‐binding of wheat bran is not related to its total dietary fiber (TDF) content. Whole (W) grain as well as pearled (P) hard red winter wheat (Hrw), hard white winter wheat (Hww), and durum wheat (DU) cooked grains were evaluated for in vitro, bile‐acid‐binding relative to cholestryramine (a cholesterol lowering bile‐acid‐binding drug). On dry matter basis (db) relative bile‐acid‐binding values were 7.7% WHrw; 7.5% WHww; 6.3% PHww; 6.0% PHrw; 5.5% WDU; and 5.4% PDU. On a TDF basis, binding values were 42–57% of that for cholestyramine for the whole and pearled wheat grains tested. Bile‐acid‐binding values (db) for WHrw and WHww were similar and significantly higher than those of PHww, PHrw, WDU and PDU. Similar bile‐acid‐binding of WHww to that of WHrw suggest that the red color commonly associated with whole grain may not necessarily indicate more healthful potential. Data suggest that cooked WHrw and WHww wheat have significantly higher health‐promoting potential than pearled grains. WDU or PDU wheat health‐promoting potential was similar to that of PHww or PHrw. Consumption of products containing WHrw and WHww are recommended.     

Apoplastic pH and monolignol addition rate effects on lignin formation and cell wall degradability in maize

Monolignol polymerization rate and apoplastic pH and may influence the formation of lignin and its interactions in cell walls. Primary maize walls were artificially lignified by gradual "end-wise" or rapid "bulk" polymerization of coniferyl alcohol at pH 4 or 5.5. Lignification efficiency was greatest for end-wise polymers at pH 5.5 (90-98%), intermediate for bulk polymers formed at either pH (54-82%), and lowest for end-wise polymers at pH 4 (41-53%). End-wise polymers had about 2.2-fold more ether inter-unit linkages and 70% fewer end-groups than bulk polymers. Low pH enhanced the formation of ether linkages in end-wise but not in bulk polymers. Differences in lignin structure did not influence the enzymatic degradability of cell walls, but lowering apoplastic pH from 5.5 to 4.0 during lignification reduced cell wall degradability by 25%. Further studies indicated this pH-dependent depression in degradability was related to cell wall cross-links formed via lignin quinone methide intermediates.     

Model studies of ferulate-coniferyl alcohol cross-product formation in primary maize walls: implications for lignification in grasses

Ferulate and diferulates mediate cell wall cross-linking in grasses, but little is known about their cross-coupling reactions with monolignols and their role in lignin formation in primary cell walls. Feruloylated primary walls of maize were artificially lignified and then saponified to release ferulate and diferulates and their cross-products with coniferyl alcohol for analysis by GC-FID, GC-MS, and NMR spectroscopy. Ferulate and 5-5-coupled diferulate had a greater propensity than 8-coupled diferulates to copolymerize with coniferyl alcohol, forming mostly 4-O-beta' and 8-beta' and some 8-O-4' and 8-5' cross-coupled structures. Some 8-beta' structures de-esterified from xylans, but these cross-links were subsequently replaced as 8-coupled diferulates formed stable cross-coupled structures with lignin. Based on the incorporation kinetics of ferulate and diferulates and the predicted growth of lignin, cross-products formed at the onset of lignification acted as nucleation sites for lignin polymerization.     

Cell wall composition of smooth bromegrass plants selected for divergent fiber concentration

Neutral detergent fiber (NDF) is considered the single best laboratory predictor of voluntary intake by ruminant livestock, creating interest in using NDF as a selection criterion in forage breeding programs. Because genetic reductions in NDF lead to increases in dry matter digestibility but not to changes in digestibility of the NDF fraction, we postulated that low-NDF plants do not have altered compositions of their cell walls. We tested this hypothesis using clones of smooth bromegrass (Bromus inermis Leyss.) with divergent NDF concentrations. High-NDF and low-NDF plants did not differ in cell wall concentrations or in the concentrations of any cell wall component (fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, uronic acids, and lignin). Instead, low-NDF plants had a cell wall that was more susceptible to solubilization in neutral detergent solution, suggesting that their cell walls were less well-developed as compared to high-NDF plants. NDF should not be used as a substitute for cell wall concentration in forage plants.     

Soluble and Insoluble Dietary Fiber Content and Composition in Oat

Six oat genotypes were grown in nursery yield trials during 1989-1992 at Lisbon, ND. Groats were analyzed for soluble and insoluble dietary fiber content and composition. Genotype-by-growing year interaction was not significant for soluble or insoluble dietary fiber. Soluble and insoluble dietary fiber differed with genotype (6.0–7.1% and 4.1– 4.9%, respectively) and with growing year (6.0–6.9% and 3.9–5.2%, respectively). The genotype-by-growing year interaction was significant for soluble β-glucan content but not for total neutral sugar or uronic acid content of the soluble dietary fiber. Genotypes did vary in total neutral sugar content but not in uronic acid content. The genotype-by-growing year interaction was not significant for total neutral sugar, β-glucan, uronic acid, or Klason lignin content of insoluble dietary fiber. Genotypes did vary in total neutral sugar, β-glucan, and Klason lignin content but not in uronic acid content of insoluble dietary fiber. The neutral sugar content of soluble dietary fiber was composed of glucose, arabinose, xylose, and galactose. The neutral sugar content of insoluble fiber was composed of glucose, arabinose, and xylose. The content and composition of soluble and insoluble dietary fiber varied with oat genotype. Therefore, oat genotypes could be bred for specific dietary fiber content and composition.     

Effect of germination on the carbohydrate composition of the dietary fiber of peas (Pisum sativum L.)

The effect of different conditions of pea germination on dietary fiber (DF) composition was studied. Insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) were subjected to acid hydrolysis, and the resultant neutral sugars, uronic acids, and Klason lignin were quantified. Germinated peas exhibited significantly higher contents of total dietary fiber (TDF) than the raw sample, due to the increases of both DF fractions. Under darkness conditions, germination exhibited the highest contents of IDF and SDF. Decreasing IDF/SDF ratios showed that the carbohydrate changes did not take place to the same extent during germination, the SDF fraction being the most affected. The detailed chemical composition of fiber fractions reveals increases of cellulose in the IDF of germinated samples, whereas SDF exhibits a decrease of pectic polysaccharides and also increases of polysaccharides rich in glucose and mannose. The DF results were corroborated by a comparative examination of the cell wall carbohydrate composition.     

In Vitro Binding of Bile Acids by Rice Bran,Oat Bran,Barley and β‐Glucan Enriched Barley

The in vitro bile acid binding by rice bran, oat bran, dehulled barley, and β‐glucan enriched barley was determined using a mixture of bile acids at a duodenal physiological pH of 6.3. Six treatments and two blank incubations were conducted testing substrates on an equal protein basis. The relative in vitro bile acid binding of the cereal brans on an equal total dietary fiber (TDF) and insoluble dietary fiber (IDF) basis considering cholestyramine as 100% bound was rice bran 45 and 49%; oat bran 23 and 30%; dehulled barley 33 and 57%; and β‐glucan enriched barley 20 and 40%, respectively. Bile acid bindings on equal protein basis for the respective cereals were 68, 26, 41, and 49%. Bile acid binding by rice bran may account to a great extent for its cholesterol‐lowering properties, while bile acid binding by oat bran suggests that the primary mechanism of cholesterol lowering by oat bran is not due to the bile acid binding by its soluble fiber. Bile acid binding was not proportional to the soluble fiber content of the cereal brans tested. Except for dehulled barley, bile acid binding for rice bran, oat bran, and β‐glucan enriched barley appear to be related to their IDF content. Highest relative bile acid binding values for rice bran and β‐glucan enriched barley were observed on an equal protein basis, whereas highest values for dehulled barley were based on IDF. Data suggest that of all four cereals tested, bile acid binding may be related to IDF or protein anionic, cationic, physical and chemical structure, composition, metabolites, or their interaction with active binding sites.     

Effects of konjac glucomannan on putative risk factors for colon carcinogenesis in rats fed a high-fat diet

The aim of this study was to determine effects of konjac glucomannan (KGM) in a high fat corn oil diet on risk factors of colon carcinogenesis, that is, fecal β-glucuronidase, mucinase, and bile acids, and on preventive factors, that is, fecal microflora and cecal short-chain fatty acids (SCFAs). Sprague-Dawley rats (n = 8 animals per group) were fed a normal-fat fiber-free (5% corn oil, w/w) or high-fat (25% corn oil, w/w) diet containing no fiber, KGM (5%, w/w), or inulin (5%, w/w, as a prebiotic control) for 4 weeks. Results indicated that the high-fat fiber-free diet significantly elevated the fecal β-glucuronidase and mucinase activities and total bile acid concentration and decreased cecal SCFA contents, as compared with its normal-fat counterpart. The incorporation of KGM, as well as inulin, into the high-fat fiber-free diet beneficially reduced the fecal β-glucuronidase and mucinase activities and lithocholic acid (secondary bile acid) concentration. Although KGM elevated the daily fecal total bile acid excretion, the change was due to the primary, instead of the secondary, bile acids. In addition, KGM beneficially promoted the daily fecal excretion of bifidobacteria and lactobacilli and cecal SCFA contents, as compared with the high-fat fiber-free diet. Therefore, the present study suggests that KGM potentially attenuated the high fat-induced risk in colon carcinogenesis.     

Pectin does not inhibit intestinal carcinogenesis in APC-deficient Min/+ mice

APC-germline mutation creates predisposition for intestinal tumorigenesis. APCMin/+ mice, developing tumors preferentially in the small intestine and only minimally in the colon, were fed pectin-enriched diets (10% galacturonan; degree of methoxylation=37.0 and 70.4%) or standard diet. Pectins used in the present study do not inhibit intestinal tumorigenesis and rather accelerate it in APCMin/+ mice. Both pectins exhibited prebiotic effects associated with high fermentative formation of acetate but producing low butyrate. The differences of the short-chain fatty acid concentrations between cecum and colon and those between colon and feces were larger than expected and increased with cancer progression, indicating an inhibition of butyrate absorption. Pectins transported more bile acids toward the colon than the standard diet and caused a higher generation of secondary bile acids despite lower pH values. Overexpression of COX-2 resulted in lower antioxidative capacity, thus promoting cancer. Apoptosis increased in hyperplasia but decreased in late adenomas. When biological modular design principles are taken into consideration, it can be expected that pectin also reinforces colorectal tumorigenesis of patients suffering from APC gene defects.     

Influence of prolonged arable cropping on lignin compounds in sandy soils of the South African Highveld

The preservation of plant residues is important for sustainable arable cropping. Lignin is a marker for plant residues in soils. We have investigated influences of the length of cultivation on the dynamics of lignin. Composite samples were taken from the top 20 cm of soils that have been cropped for periods varying from 0 to 98 years in each of three different agro‐ecosystems in the Free State Province of South Africa. Lignin‐derived phenols were determined in the <2 µm (clay), 2–20 µm (silt), 20–250 µm (fine sand) and 250– 2000 µm (coarse sand) size separates. With increasing length of cultivation, the concentration of such phenols decreased to 36% of that in the grassland. The lignin contents as proportions of the total carbon did not change during cultivation, suggesting that there was no selective enrichment of lignin moieties as C was lost as a result of cultivation. The loss rate constants of lignin concentrations in particle‐size fractions increased in the order clay (0.17 year^?1) ≤ silt (0.18 year^?1) < fine sand (0.20 year^?1) < coarse sand (0.22 year^?1). Increasing ratios of phenolic acids to aldehydes in bulk soil, silt and fine sand fractions with increasing length of cultivation indicated that side chains were being oxidized. The ratios in the silt fraction, however, decreased after 10–20 years. We attribute this to a loss of lignin together with silt by wind erosion, resulting in a rejuvenation of lignin compounds in the remaining silt‐sized pools of C.     

Retention of coumaric acid by soil and its colloidal components

In this study of the behavior of coumaric acid added to soil, the disappearance of the acid was found to be due to adsorption plus microbial degradation, and was influenced by the concentration and contact time. Adsorption experiments set up with soils varying widely in their chemical and physical properties, showed that the Freundlich isotherm fits the data quite well and that among the different soil factors, only the pH was closely and negatively correlated with coumaric acid adsorption. Approximately at pH > 7 no adsorption occurred, perhaps due to the repulsion between the negatively charged soil colloids and the dissociated acidic groups of coumaric acid. Adsorption experiments carried out with different adsorbents showed that the hydroxy-Fe compound was the most effective in retaining coumaric acid, followed by humic acid, illite, kaolinite and vermiculite, in this order.     

Changes in herbage chemical composition due to proportion of species in alfalfa‐orchardgrass mixtures

Abstract

Legumes and grasses are widely grown in mixtures throughout the United States and Canada. This study was conducted to determine the changes in chemical composition of herbage that occur as proportion of legume and grass change in the mixture. Composition of mixtures of alfalfa (Medicago sativa L.) and orchardgrass (Dactylis glomerata L.) were studied after their herbage had been mixed by weight into seven different proportions: 100% alfalfa ‐ 0% orchardgrass, 80% ‐ 20%, 65% ‐ 35%, 50% ‐ 50%, 35% ‐ 65%, 20% ‐ 80%, and 0% ‐ 100%, respectively. The forages were grown in rows of pure alfalfa or pure orchardgrass spaced 45 cm apart on fertilized Dodge silt loam (Typic Hapludalf). Alfalfa was at late bud and orchardgrass was a few days from the appearance of the first anthers when harvested on May 29, 1975. Four replications of each of the mixtures were prepared, and the mixtures were made after tissues had been ground to 40‐mesh size.

No significant differences among mixtures were found for S, Cu, total nonstructural carbohydrates (TNC), or in vitro digestible dry matter (IVDDM). Concentrations of N, P, Ca, Mg, Na, Al, Ba, Fe, Sr, B, Zn, starch, and acid‐detergent lignin (ADL) were highest in pure alfalfa herbage and significantly decreased in concentration as amount of orchardgrass in the mixture increased. In direct contrast, concentrations of K, Mn, total sugars, fructosan, cell wall constituents (CWC), cellulose, hemicellulose, and acid detergent fiber (ADF) were lowest in pure alfalfa herbage and significantly increased in concentration as amount of orchardgrass in the mixture increased. These data show that an increase in amount of orchardgrass in a mixture with alfalfa reduced the concentrations of most herbage constituents important to animal nutrition and increased the fibrous constituents.