Solati Z., Jørgensen U. and Søegaard K.
Aarhus University, Department of Agroecology, Blichers allé 20, 8830 Tjele, Denmark;
zeinab.solati@agro.au.dk
Abstract
Legume and grass crops are potential alternative protein sources to substitute the high levels of importation of soybean meal for animal production. The quantities of crude protein fractions were estimated in legumes, namely white clover (Trifolium repens L.), red clover (Trifolium pratense L.) and lucerne (Medicago sativa L.) and grass species, namely perennial ryegrass (Lolium perenne L.) and tall fescue (Festuca arundinacea L.) across six harvest dates during spring growth, using the Cornell net carbohydrate and protein system. The production of crude protein fractions was strongly affected by species. Red clover showed the highest B3 fraction productivity and true protein extractability across the harvest dates compared with other species. White clover and red clover demonstrated higher production of B2 fraction in early growth compared with other species.
Keywords: CNCPS, protein fractions, spring growth, morphological development, legume, grass
Introduction
European countries are importing large quantities of protein mainly in the form of soybean meal for animal production. The extent of feed proteins imported into the EU has raised various concerns such as susceptibility of animal production to price volatility, deforestation, biodiversity loss, and soil and water pollution (Van Krimpen et al., 2013) which indicates the importance of identifying alternative protein sources within the EU to substitute for the importation of soybean. The quantity, quality and price of the alternative protein should be comparable to that of soybean meal if substitution is to be successful. Legumes and grasses are important sources of protein due to the high quantity of up to 250 g kg-1 dry matter (DM) (Lyttelton, 1973). Moreover, the nutritional quality of leaf protein was shown to be comparable to that of soy protein (Pirie, 1987). If leaf protein production can be integrated with a biorefinery which produces other products, the overall economics could be considerably improved (Dale et al., 2009). Therefore, biorefinery of grasses and legume crops for production of protein concentrates for animals is an interesting option. In this study, protein fractions were estimated according to the Cornell Net Carbohydrate and Protein System (CNCPS) which fractionates the crude protein into five different fractions. It was assumed that the protein fractions estimated by CNCPS correlate with the extractability of protein fractions in a biorefinery. The objective of this study was to estimate the protein fractions of legume and grass species during the spring growth.
Materials and methods
The legume (white clover, red clover, lucerne) and grass (perennial ryegrass and tall fescue) species were harvested during the spring growth on 6 different dates: 11 May, 19 May, 26 May, 1 June, 8 June and 16 June. Plots were established in 2014 and harvested in 2015. Grasses were fertilized in spring 2015 with 140 kg N ha-1 and legumes were unfertilized. Field plots were arranged according to a split plot design with four replications. Samples were analyzed for crude protein fractions according to CNCPS into five different fractions using the methodology of Licitra et al. (1998). Fraction A was estimated using trichloroacetic acid (TCA) as protein precipitant agent and determination of total Kjeldahl nitrogen in the precipitate. Crude protein fraction B1 was determined as borate-phosphate buffer insoluble protein fraction subtracted from TCA precipitated protein. Fraction B3 was assessed as acid detergent insoluble
protein (ADIP) deducted from neutral detergent insoluble protein (NDIP). Fraction C was estimated as ADIP, and finally fraction B2 was estimated by deducting NDIP from the buffer insoluble protein fraction. Effect of species and morphological development on the quantity of protein fractions were evaluated according to the mixed model using R statistical software. Plant species and morphological development were fixed variables while field replication was random variable. Pairwise comparisons were performed using the Tukey HSD. A probability of P<0.05 was declared significant.
Results and discussion
The amount of each protein fraction of legume and grass species across harvest dates is presented in Figure 1. Plant species and morphological development influenced the productivity of crude protein fractions.
Assuming fraction C is not extractable and fraction B is extractable, the highest extractable true protein (B1 + B2 + B3) was determined for red clover at most of the harvest dates. This could be attributed to the significantly higher (P<0.05) amount of B3 fraction compared with other species. The highest extractable true protein for red clover was achieved at the 4th harvest date with a value of 726 kg ha-1 after which there was a decline in the amount of extractable true protein. At the second harvest date, white clover demonstrated extractable true protein content (404 kg ha-1) which was significantly (P<0.05) higher than that of all other species except red clover. The highest extractable true protein content for white clover was achieved at the 5th harvest date with the value of 535 kg ha-1. Grass species contained the lowest extractable true protein at the first harvest date and the value increased (P<0.05) from first to the last harvest date. Extractable true protein increased from 221 to 478 kg ha-1 for perennial ryegrass and from 202 to 449 kg ha-1 for tall fescue from first to the last harvest date.
The extractability of true protein, meaning B1 + B2 + B3 was based on the assumption that B3 fraction which is bound to the cell wall might become extractable using enzymes. However, if B3 is not extractable, true protein can be defined as B1 + B2 fractions which was shown to be significantly higher (P<0.05)
0 50 100 150 200 250 300 350 400 450 500
11 May 19 May 26 May 1 June 8 June 16 June 11 May 19 May 26 May 1 June 8 June 16 June 11 May 19 May 26 May 1 June 8 June 16 June 11 May 19 May 26 May 1 June 8 June 16 June 11 May 19 May 26 May 1 June 8 June 16 June
WC RC LU PR TF
A B1 B2 B3 C
CP fractions (kg ha-1 )
Figure 1. Amount of individual CP fractions (kg ha-1) for legume (WC= white clover, RC= red clover, LU= lucerne) and grass (PR= perennial ryegrass, and TF= tall fescue) species across the harvest dates during the spring growth. Bars indicate the standard error of the mean. Error bars that are smaller than the width of the symbol are not visible.
for white clover (265 kg ha-1) and red clover (332 kg ha-1) only at the first harvest date (early growth) compared with other species. McKenzie (1977) reported protein yields of 75 and 77 kg ha-1 on May 2 and June 6 respectively for white clover which was achieved using a laboratory scale pulper and press.
Irrespective of the fact that our results only represent an estimation of protein extractability in a biorefinery, the discrepancy in the quantity of extractable protein (kg ha-1) can be attributed to differences in the DM yield and protein extraction condition in general; as alkaline pH, warm aqueous extraction solutions and pretreatment (disruption of cell walls) benefit the protein extraction (Dale et al., 2009) which were not incorporated in McKenzie’s (1977) study.
Generally, the B2 fraction exhibited the highest and fraction C the lowest amount in all species. There was no significant difference in the quantity of fraction B2 between grass species at any harvest date.
Moreover, grasses and legumes did not differ significantly in B2 fraction content at the 3rd and last harvest dates. However, the amount of B2 fraction was higher (P<0.05) for white clover and red clover at the first two harvest dates compared with other species. The amount of fraction B1 did not differ among the species at any harvest date.
Conclusions
The results showed that the amount of crude protein fractions can be manipulated by selection of both morphological development stage and plant species. We anticipate that the estimation of crude protein fractions can provide knowledge of the suitability of the crops for protein production in an integrated biorefinery system. However, it is not yet determined how well the actual extractability of different crude protein fractions correlates with CNCPS crude protein estimation. This will require further investigation of parallel physical extraction and CNCPS estimation.
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