A modified rinsing method for the determination of the S, W-S and D + U fraction of protein and starch in feedstuff within the in situ technique.

A modified rinsing method for the in situ technique was developed to separate, isolate and characterise the soluble (S), the insoluble washout (W-S) and the non-washout fractions (D + U) within one procedure. For non-incubated bags (t = 0 h), this method was compared with the conventional, Combined Fractionation (CF) method that measures the D + U and S fractions in separate steps and subsequently calculates the W-S fraction. The modified method was based on rinsing of nylon bags in a closed vessel containing a buffer solution (pH 6.2) during 1 h, where shaking speeds of 40, 100, and 160 strokes per minutes (spm) were evaluated, and tested for six feed ingredients (faba beans, maize, oats, peas, soya beans and wheat) and four forages (two ryegrass silages and two maize silages). The average recoveries as the sum of all fractions were 0.972 ± 0.041 for N and 0.990 ± 0.050 for starch (mean ± s.d.). The mean W-S fraction increased with increasing shaking speed and varied between 0.017 (N) and 0.083 (starch) at 40 spm and 0.078 (N) and 0.303 (starch) at 160 spm, respectively. For ryegrass silages, the W-S fraction was absent at all shaking speeds, but was present in the CF method. The modified method, in particular at 40 and 100 spm, reduced the loss of small particles during rinsing, resulting in lower W-S and higher D + U fractions for N and starch compared with the CF method. For soya beans and ryegrass silage, the modified method reduced the S fraction of N compared with the CF method. The results obtained at 160 spm showed the best comparison with those from the CF method. The W-S fraction of the feedstuff obtained at 160 spm contained mainly particles smaller than 40 μm (0.908 ± 0.086). In most feedstuff, starch was the most abundant chemical component in the W-S fraction and its content (726 ± 75 g/kg DM) was higher than in the D + U fraction (405 ± 177 g/kg DM). Alkaline-soluble proteins were the dominant N-containing components in the W-S fraction of dry feed ingredients and its relative content (0.79 ± 0.18 of total N in W-S) was higher than in the D + U fraction (0.59 ± 0.07 of total N in D + U) for all feedstuff except maize. The molecular weight distribution of the alkaline-soluble proteins differed between the W-S and the D + U fractions of all dry feed ingredients, except soya beans and wheat.