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United States Department of Agriculture

Agricultural Research Service

Title: Utilizing the Acetyl Bromide Assay to Determine Lignin Concentrations in Herbaceous Plants: Some Cautionary Notes

Authors
item Hatfield, Ronald
item Grabber, John - MONSANTO-PROTIVA
item Ralph, John
item Brei, K - UNIV OF WISCONSIN-MADISON

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 3, 1997
Publication Date: N/A

Interpretive Summary: Forages play a critical role in providing nutrients to ruminants. Structural carbohydrates that make up the bulk of forage fiber are poorly digested by ruminants. It has generally been observed that as lignin (an indigestible fiber component) increases in forages structural carbohydrate digestion decreases. Although this observation is well established, the mechanism behind the decreased digestion is unknown. Accurate determinatio of lignin content of forages is critical to understanding the relationship between lignin and structural carbohydrate digestibility. Once we understand this relationship, a simple estimation of lignin concentration may allow us to predict the digestibility and nutrient value of a given forage. The acetyl bromide assay for lignin is a simple and straightforward method that does not require large sample sizes. However, recent modifications using perchloric acid to aid dissolving the fiber led to an over estimation of lignin due to specific degradation of xylans. Xylans ar a type of structural carbohydrate found in all types of forage fiber. We have found that lowering the reaction temperature and omitting the perchloric acid minimizes interference from xylan degradation while providing reliable and accurate lignin determinations in forage samples. This method may prove especially useful for accurately determining the lignin content of plants that have been bioengineered to contain less lignin or a modified lignin composition. The acetyl bromide method is easily adapted to small sample size, allows large numbers of assays, and should accurately estimate lignin values even though lignin composition may have significantly changed. This method has much greater flexibility than current lignin determination methods.

Technical Abstract: The acetyl bromide assay was developed to provide a rapid and sensitive method for quantifying lignin in woody plant species. The original procedure cautioned against prolonged reaction times and advised keeping the reaction temperature at 70 deg C to prevent excessive carbohydrate degradation that would skew the absorption spectra. Characterization of the ereaction conditions revealed that the acetyl bromide reagent readily degrades xylans, a prominent polysaccharide group within all lignified plants. This degradation results in increased absorbance in the 270 to 280 nm region that is used to quantify lignin. The degradation of xylans is temperature dependent and is exacerbated by the addition of perchloric acid. Lowering the reaction temperature to 50 deg C and increasing the reaction time from 2-4 h allows complete lignin solubilization but minimizes degradation of the xylans.

Last Modified: 9/10/2014
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