The acetyl bromide lignin method accurately quantitates lignin in forage
Introduction
Lignin, a phenolic biopolymer, is a component of cell walls in plants that resists digestion by ruminant animals, limiting their value as an energy source (Van Soest, 1994; Hatfield and Fukushima, 2005). Understanding the biological processes required for cell wall polysaccharide degradation and the limitations on digestion imposed by lignin requires that lignin be defined chemically (Faichney, 1975). There are numerous methods to determine lignin in forages, but there is no single accepted analytical method. The lignin (sa) acid detergent lignin (ADL) method (Van Soest, 1963), an alternative to another sulfuric acid procedure (Klason lignin), is commonly used in animal science because protein is sometimes found in the fibrous substrate used in the Klason lignin method. To avoid such interference, Van Soest (1963) developed acid detergent fiber (ADF), including a detergent to remove protein, and since then ADL has been used extensively to quantitate lignin in forage plants. However, there is some concern that the ADL method underestimates lignin, primarily in Gramineae species, due to lignin’s solubility in the acid detergent solution used to obtain ADF (Shimojo and Goto, 1984; Kondo et al., 1987; Lowry et al., 1994).
The spectrophotometric acetyl bromide lignin (ABL) method employed for determining lignin in lumber (Johnson et al., 1961) and forages (Morrison, 1972a,b) uses as the fibrous substrate crude cell wall (CW), that is obtained treating sequentially the ground forage with water, ethanol, choroform:methanol (2:1) and acetone in a Soxhlet extractor. Lignin concentration, based on its dissolved phenolic core, is calculated spectrophotometrically at OD280 (Johnson et al., 1961; Iiyama and Wallis, 1989). Tannins, flavonoids, protein, etc. that may also absorb at this wavelength are either washed away during the CW preparation or are insoluble in the acetyl bromide reagent (Morrison, 1972a,b). The acetyl bromide method is a suitable analytical tool to determine lignin concentrations in herbaceous tissues (Moreira-Vilar et al., 2014). We have previously compared the spectrophotometric ABL procedure with the gravimetric ADL method (Fukushima et al., 2015a). Here, we extend that work to demonstrate, using the degradability of forage versus the lignin content, that the higher recovery of lignin in grasses in the ABL method, compared to the ADL, may make it more useful for predicting degradability and thus nutritive value of forage.
Section snippets
Plant materials, methods of analysis, and statistical analyses
The data used in this study came primarily from our previous research; therefore, details on plant materials, methods and their analysis, in vitro forage degradability assays, and statistical analyses are described in that study (Fukushima et al., 2015a). In the previous study, lignin data were standardized only on the basis of the amount of aNDF; however, here lignin data are standardized on the basis of DM and aNDF. For the degradability assays, anaerobiosis of the buffer solution, using
Lignin concentrations
Our previous work (Fukushima et al., 2015a) reported lignin data (as well as correlation coefficients) standardized only for aNDF. In this work, data for the lignin values (g/kg DM and g/kg aNDF) obtained through the two methods and the forage degradability data that were used for linear regression analysis are presented in Table 1, Table 2. The ABL values in grasses were higher (P < 0.0001) than the corresponding ADL values (58 vs. 27 g/kg DM and 106 vs. 56 g/kg aNDF, respectively), with
Lignin concentrations
Low ADL yields reported in the literature are probably due to the solubilization and loss of lignin during the ADL procedure, especially in tropical grasses where up to 50 % is lost (Shimojo and Goto, 1984; Lowry et al., 1994; Hatfield et al., 1994; Jung et al., 1997; Moore and Jung, 2001). Jung et al. (1999) suggested that the ADL process underestimated lignin when they found that the amount of lignin recovered was less than the gross energy determined by bomb calorimetry. We also found low
Conclusions
Based on the results presented here, we made several conclusions.
- 1
The ABL method shows that forage degradability as a function of lignin concentration is similar for grasses and legumes and suggests that the lignin-mediated inhibition of enzymatic digestion of cell wall polysaccharides is similar for both types of plants.
- 2
The lower degradability of ADL in grasses versus legumes is due to a partial loss of lignin in grasses during the processing of the samples. This apparent discrepancy in
Declaration of Competing Interest
The authors declare that they have no conflicts of interest.
CRediT authorship contribution statement
R.S. Fukushima: Conceptualization, Methodology, Investigation, Writing - original draft, Writing - review & editing. M.S. Kerley: Supervision, Resources, Project administration. M.H. Ramos: Methodology, Software. R.L. Kallenbach: Resources, Validation, Writing - review & editing, Funding acquisition.
Acknowledgments
Recognition is due to “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – grant number BEX 3711/07-2” and “Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) – grant number 2009/00074-9” - Brazil, for the fellowships awarded to the corresponding author.
Thanks to Mr. James H. Porter for his laboratory expertise.
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